Porous beta-tricalcium phosphate granules and methods for producing same

ABSTRACT

A porous β-tricalcium phosphate material for bone implantation is provided. The multiple pores in the porous TCP body are separate discrete voids and are not interconnected. The pore size diameter is in the range of 20-500 μm, preferably 50-125 μm. The porous β-TCP material provides a carrier matrix for bioactive agents and can form a moldable putty composition upon the addition of a binder. Preferably, the bioactive agent is encapsulated in a biodegradable agent. The invention provides a kit and an implant device comprising the porous β-TCP, and a bioactive agent and a binder. The invention also provides an implantable prosthetic device comprising a prosthetic implant having a surface region, a porous β-TCP material disposed on the surface region and optionally comprising at least a bioactive agent or a binder. Methods of producing the porous β-TCP material and inducing bone formation are also provided.

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 09/798,518, filed Mar. 2, 2001.

BACKGROUND OF THE INVENTION

[0002] Bone tissue in the human body comprises the largest proportion ofthe body's connective tissue mass. However, unlike other connectivetissues, its matrix consists of physiologically mineralized, tinycrystallites of a basic, carbonate-containing calcium phosphate calledhydroxyapatite distributed in an organized collagen structure. Repair ofthis tissue is a complex process involving a number of cellularfunctions directed towards the formation of a scaffold andmineralization of the defect followed by an eventual remodeling of thedefect site to attain the original structure.

[0003] Implantations of calcium phosphate based biomaterials have beenfound to be generally compatible and conducive to bone repair. Bonerepair is influenced by a number of physico-chemical variablesassociated with calcium phosphate such as the calcium to phosphate molarratio. Hydroxyapatite and tricalcium phosphate are widely used in boneimplants. Hydroxyapatite has the chemical formula Ca₁₀(PO₄)₆(OH)₂, andthe ratio of calcium to phosphate is about 1.67. Tricalcium phosphate(TCP) has the formula of Ca₃(PO₄)₂, and the ratio of calcium tophosphate is about 1.5. Tricalcium phosphate has biological propertiesof being non-reactive and resorbable. It acts as a scaffolding for boneingrowth and undergoes progressive degradation and replacement by bone(Lange et al., Annals of Clinical and Laboratory Science, 16, pp.467-472 (1986)). TCP is degraded 10-20 times faster than hydroxyapatite.A TCP implant generally results in superior remodeling thanhydroxyapatite during the final stage of bone formation. It isnoteworthy that TCP is resorbed by osteoclast cells, whereas, the muchslower resorption of hydroxyapatite is effected mainly by foreign-bodygiant cells. The giant cells have a limit as to the amount ofhydroxyapatite they will resorb. [0004] Porous ceramic material is oftenselected as the matrix for bone implants. When such material is embeddedat the implant site, the porous material is resorbed by osteolytic cellswhich infiltrate the pores. Simultaneously, the bone tissue isregenerated by osteoblasts. A certain pore size is required forosteoblasts to invade the pore of the implant material. Parameters suchas crystallinity, solubility, particle size, porosity, pore structureand pore size of the implanted material can greatly influence bonecompatibility and bone integration. An inappropriate combination of theabove parameters can lead to improper bone repair.

[0004] The use of porous ceramics having interconnected pores as animplantable solid material for bone substitutes has been described (see,e.g., U.S. Pat. No. 5,171,720; see also Frayssinet et al., Biomaterials,14, pp. 423-429 (1993)). Such porous ceramics, however, are brittle andare not capable of being easily shaped by the practitioner during anoperation.

[0005] Excessively large pore size and high porosity of the ceramicmaterial can lead to excessive resorption rates, thus, preventing thematrix from providing a scaffold for the newly synthesized bone. Whenthe rate of resorption is faster than the rate of bone growth, it oftenleads to an inflammatory response. Small pore size and low porosity ofthe ceramic material will lead to low resorption rates causingencapsulation of matrix particles in the new bone.

[0006] It would thus be desirable to identify a biomaterial which can beapplied to a defect site and which can greatly enhance the regenerativeprocess, particularly when used with other bioactive agents such as bonemorphogenic proteins and other related factors. In addition, it would bedesirable to identify and use a matrix which acts as a mechanicallydurable carrier for the bioactive agents and is a well-tolerated bonereplacement material that favors healing.

SUMMARY OF THE INVENTION

[0007] The present invention solves these problems by identifying aporous ceramic material having a composition, pore size, porosity andgranule size for improving the regeneration of bone tissue in a livingbody, and repairing a bone defect in a human or animal. The presentinvention provides a porous β-tricalcium phosphate (β-TCP) material foruse in bone implant applications. The invention provides porous forms ofβ-TCP granules which are biocompatible and support the development ofnew bone throughout its structural form.

[0008] The invention also provides a composition comprising the porousβ-TCP with a bioactive agent such as an antibiotic, a bone morphogenicprotein (BMP), or a nucleic acid molecule comprising a sequence encodingBMP in the presence or absence of a morphogenic protein stimulatoryfactor (MPSF) to improve osteoconductivity. In a preferred embodiment,the bioactive agent is encapsulated in a biodegradable agent.Preferably, the particle size of the biodegradable agent is 20-500 μm.The porous β-TCP material or porous β-TCP/bioactive agent mixture canalso be used in conjunction with binders to form a moldable puttycomposition ready for shaping in the implant site. The invention alsoprovides a kit comprising the porous β-TCP, and at least one or moreadditional components including a bioactive agent and a binder.

[0009] In another aspect, the invention also provides an implantabledevice comprising the porous β-TCP material, and optionally comprisingone or more additional components including a bioactive agent such as aBMP, an antibiotic or a binder. The invention also provides animplantable prosthetic device comprising the porous β-TCP material andoptionally comprising one or more additional components including abioactive agent such as a BMP, an antibiotic or a binder. The prostheticdevice or implantable device comprising the porous β-TCP and BMP mayoptionally comprise a MPSF.

[0010] Another object of the invention is to provide a method ofproducing the porous β-TCP material. The method comprises blending theTCP powder with a pore-forming agent, adding a granulating solution toform a crumbly mass, passing the crumbly mass through a sieve to formgranules and sintering the granules to form the porous β-TCP.

[0011] The invention also provides a method of inducing bone formationin a mammal comprising the step of implanting in the defect site of amammal a composition comprising the porous β-TCP and optionally a binderand/or a bioactive agent. The invention describes a method of deliveringa bioactive agent at a site requiring bone formation comprisingimplanting at the defect site of a mammal a composition comprising theporous β-TCP and a bioactive agent, wherein the bioactive agent isoptionally encapsulated in a biodegradable agent. The invention alsodescribes a method of delivering a bioactive agent to a site requiringcartilage formation comprising implanting at the defect site of a mammala composition comprising the bioactive agent and biodegradable agent,wherein the bioactive agent is encapsulated in the biodegradable agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1. Histologic image of animal number 297L (left tibia) at 4weeks with placebo. From top to bottom, the sites are proximal, middleand distal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCPputty 89F, respectively.

[0013]FIG. 2. Histologic image of animal number 297R (right tibia) at 4weeks with placebo. From top to bottom, the sites are proximal, middleand distal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

[0014]FIG. 3. Histologic image of animal number 295L (left tibia) at 4weeks with OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing collagen 48C, β-TCP putty 89A, β-TCP putty 89B,respectively.

[0015]FIG. 4. Histologic image of animal number 295R (right tibia) at 4weeks with OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89C, β-TCP putty 89F, control,respectively.

[0016]FIG. 5. Histologic image of animal number 299L (left tibia) at 8weeks with placebo. From top to bottom, the sites are proximal, middleand distal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCPputty 89F, respectively.

[0017]FIG. 6. Histologic image of animal number 299R (right tibia) at 8weeks with placebo. From top to bottom, the sites are proximal, middleand distal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

[0018]FIG. 7. Histologic image of animal number 138L (left tibia) at 8weeks with OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89A, β-TCP putty 89B, β-TCP putty89C, respectively.

[0019]FIG. 8. Histologic image of animal number 138R (right tibia) at 8weeks with OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89F, control, collagen 48C,respectively.

[0020]FIG. 9. Radiographic image of animal number 297L (left tibia) at 4weeks with placebo. From the right, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

[0021]FIG. 10. Radiographic image of animal number 297R (right tibia) at4 weeks with placebo. From the left, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

[0022]FIG. 11. Radiographic image of animal number 295L (left tibia) at4 weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing collagen 48C, β-TCP putty 89A, β-TCP putty 89B,respectively.

[0023]FIG. 12. Radiographic image of animal number 295R (right tibia) at4 weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing β-TCP putty 89C, β-TCP putty 89F, control,respectively.

[0024]FIG. 13. Radiographic image of animal number 299L (left tibia) at8 weeks with placebo. From the right, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

[0025]FIG. 14. Radiographic image of animal number 299R (right tibia) at8 weeks with placebo. From the left, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

[0026]FIG. 15. Radiographic image of animal number 138L (left tibia) at8 weeks with OP-1. From the right, the sites are proximal, middle anddistal, each containing β-TCP putty 89A, β-TCP putty 89B, β-TCP putty89C, respectively.

[0027]FIG. 16. Radiographic image of animal number 138R (right tibia) at8 weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing β-TCP putty 89F, control, collagen 48C,respectively.

[0028]FIG. 17. Paraffin scanning image of animal number 297L (lefttibia) at 4 weeks with placebo. From the top, the sites are proximal,middle and distal, each containing β-TCP putty 89B, β-TCP putty 89C,β-TCP putty 89F, respectively.

[0029]FIG. 18. Paraffin scanning image of animal number 297R (righttibia) at 4 weeks with placebo. From the top, the sites are proximal,middle and distal, each containing control, collagen 48C, β-TCP putty89A, respectively.

[0030]FIG. 19. Paraffin scanning image of animal number 295L (lefttibia) at 4 weeks with OP-1. From the top, the sites are proximal,middle and distal, each containing collagen 48C, β-TCP putty 89A, β-TCPputty 89B, respectively.

[0031]FIG. 20. Paraffin scanning image of animal number 295R (righttibia) at 4 weeks with OP-1. From the top, the sites are proximal,middle and distal, each containing β-TCP putty 89C, β-TCP putty 89F,control, respectively.

[0032]FIG. 21. Paraffin scanning image of animal number 299L (lefttibia) at 8 weeks with placebo. From the top, the sites are proximal,middle and distal, each containing β-TCP putty 89B, β-TCP putty 89C,β-TCP putty 89F, respectively.

[0033]FIG. 22. Paraffin scanning image of animal number 299R (righttibia) at 8 weeks with placebo. From the top, the sites are middle anddistal, each containing collagen 48C and β-TCP putty 89A, respectively.

[0034]FIG. 23. Paraffin scanning image of animal number 138L (lefttibia) at 8 weeks with OP-1. From the top, the sites are proximal,middle and distal, each containing β-TCP putty 89A, β-TCP putty 89B,β-TCP putty 89C, respectively.

[0035]FIG. 24. Paraffin scanning image of animal number 138R (righttibia) at 8 weeks with OP-1. From the top, the sites are proximal,middle and distal, each containing β-TCP putty 89F, control, collagen48C, respectively.

[0036]FIG. 25. Specimen 295L middle site showing one of the five poreswith bone growth, where EP is an empty pore and FP is a filled pore.

[0037]FIG. 26. Specimen 299L distal site showing 7 or 8 pores with bonegrowth, where EP is any empty pore and FP is a filled pore.

[0038]FIG. 27. Radiographic image of animal number 5333L (left tibia) at4 weeks with OP-1 encapsulated in PLGA. From the left, the sites areproximal, middle and distal, each containing control, formulation 5 andformulation 4, respectively.

[0039]FIG. 28. Radiographic image of animal number 5335L (left tibia) at8 weeks with OP-1 encapsulated in PLGA. From the left, the sites areproximal, middle and distal, each containing control, formulation 4 andformulation 5, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0040] In order that the invention herein described may be fullyunderstood, the following detailed description is set forth.

[0041] “Amino acid sequence homology” is understood to include bothamino acid sequence identity and similarity. Homologous sequences shareidentical and/or similar amino acid residues, where similar residues areconservative substitutions for, or “allowed point mutations” of,corresponding amino acid residues in an aligned reference sequence.Thus, a candidate polypeptide sequence that shares 70% amino acidhomology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Certain particularly preferred morphogenic polypeptides share at least60%, and preferably 70% amino acid sequence identity with the C-terminal102-106 amino acids, defining the conserved seven-cysteine domain ofhuman OP-1, BMP-2, and related proteins.

[0042] Amino acid sequence homology can be determined by methods wellknown in the art. For instance, to determine the percent homology of acandidate amino acid sequence to the sequence of the seven-cysteinedomain, the two sequences are first aligned. The alignment can be madewith, e.g., the dynamic programming algorithm described in Needleman etal., J. Mol. Biol., 48, pp. 443 (1970), and the Align Program, acommercial software package produced by DNAstar, Inc. The teachings byboth sources are incorporated by reference herein. An initial alignmentcan be refined by comparison to a multi-sequence alignment of a familyof related proteins. Once the alignment is made and refined, a percenthomology score is calculated. The aligned amino acid residues of the twosequences are compared sequentially for their similarity to each other.Similarity factors include similar size, shape and electrical charge.One particularly preferred method of determining amino acid similaritiesis the PAM250 matrix described in Dayhoff et al., Atlas of ProteinSequence and Structure, 5, pp. 345-352 (1978 & Supp.), which isincorporated herein by reference. A similarity score is first calculatedas the sum of the aligned pairwise amino acid similarity scores.Insertions and deletions are ignored for the purposes of percenthomology and identity. Accordingly, gap penalties are not used in thiscalculation. The raw score is then normalized by dividing it by thegeometric mean of the scores of the candidate sequence and theseven-cysteine domain. The geometric mean is the square root of theproduct of these scores. The normalized raw score is the percenthomology.

[0043] “Biocompatible” refers to a material that does not elicitdetrimental effects associated with the body's various protectivesystems, such as cell and humoral-associated immune responses, e.g.,inflammatory responses and foreign body fibrotic responses. The termbiocompatible also implies that no specific undesirable cytotoxic orsystemic effects are caused by the material when it is implanted intothe patient.

[0044] “Binder” refers to any biocompatible material which, when admixedwith osteogenic protein and/or the porous matrix promotes boneformation. Certain preferred binders promote such repair using lessosteogenic protein than standard osteogenic devices. Other preferredbinders can promote repair using the same amount of the osteogenicprotein as the standard osteogenic devices while some require more topromote repair. As taught herein, the skilled artisan can determine aneffective amount of protein for use with any suitable binder using onlyroutine experimentation. Among the other characteristics of a preferredbinder is an ability to render the device: pliable, shapeable and/ormalleable; injectable; adherent to bone, cartilage, muscle and othertissues, resistant to disintegration upon washing and/or irrigatingduring surgery; and, resistant to dislodging during surgery, suturingand post-operatively, to name but a few. Additionally, in certainpreferred embodiments, a binder can achieve the aforementioned featuresand benefits when present in low proportions.

[0045] “Biodegradable agent” refers to a resorbable biocompatiblematerial such as a material that degrades gradually at the implant site.The material is capable of encapsulating a bioactive agent to providetime release or sustained release delivery of the bioactive agent. Thebiodegradable material encompasses natural and synthetic polymers.Examples of biodegradable material are poly(L-lactide) (PLLA),poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA)and co-polymers thereof.

[0046] “Bone” refers to a calcified (mineralized) connective tissueprimarily comprising a composite of deposited calcium and phosphate inthe form of hydroxyapatite, collagen (primarily Type I collagen) andbone cells such as osteoblasts, osteocytes and osteoclasts, as well asto bone marrow tissue which forms in the interior of true endochondralbone. Bone tissue differs significantly from other tissues, includingcartilage tissue. Specifically, bone tissue is vascularized tissuecomposed of cells and a biphasic medium comprising a mineralized,inorganic component (primarily hydroxyapatite crystals) and an organiccomponent (primarily of Type I collagen). Glycosaminoglycans constituteless than 2% of this organic component and less than 1% of the biphasicmedium itself, or of bone tissue per se. Moreover, relative to cartilagetissue, the collagen present in bone tissue exists in a highly-organizedparallel arrangement. Bony defects, whether from degenerative, traumaticor cancerous etiologies, pose a formidable challenge to thereconstructive surgeon. Particularly difficult is reconstruction orrepair of skeletal parts that comprise part of a multi-tissue complex,such as occurs in mammalian joints.

[0047] “Bone formation” means formation of endochondral bone orformation of intramembranous bone. In humans, bone formation beginsduring the first 6-8 weeks of fetal development. Progenitor stem cellsof mesenchymal origin migrate to predetermined sites, where they either:(a) condense, proliferate, and differentiate into bone-forming cells(osteoblasts), a process observed in the skull and referred to as“intramembranous bone formation” or, (b) condense, proliferate anddifferentiate into cartilage-forming cells (chondroblasts) asintermediates, which are subsequently replaced with bone-forming cells.More specifically, mesenchymal stem cells differentiate intochondrocytes. The chondrocytes then become calcified, undergohypertrophy and are replaced by newly formed bone made by differentiatedosteoblasts, which now are present at the site. Subsequently, themineralized bone is extensively remodeled, thereafter becoming occupiedby an ossicle filled with functional bone-marrow elements. This processis observed in long bones and referred to as “endochondral boneformation.” In postfetal life, bone has the capacity to repair itselfupon injury by mimicking the cellular process of embryonic endochondralbone development. That is, mesenchymal progenitor stem cells from thebone-marrow, periosteum, and muscle can be induced to migrate to thedefect site and begin the cascade of events described above. There, theyaccumulate, proliferate, and differentiate into cartilage, which issubsequently replaced with newly formed bone.

[0048] “Bone morphogenic protein (BMP)” refers to a protein belonging tothe BMP family of the TGF-β superfamily of proteins (BMP family) basedon DNA and amino acid sequence homology. A protein belongs to the BMPfamily according to this invention when it has at least 50% amino acidsequence identity with at least one known BMP family member within theconserved C-terminal cysteine-rich domain which characterizes the BMPprotein family. Members of the BMP family may have less than 50% DNA oramino acid sequence identity overall.

[0049] “Conservative substitutions” are residues that are physically orfunctionally similar to the corresponding reference residues. That is, aconservative substitution and its reference residue have similar size,shape, electric charge, chemical properties including the ability toform covalent or hydrogen bonds, or the like. Preferred conservativesubstitutions are those fulfilling the criteria defined for an acceptedpoint mutation in Dayhoff et al., supra. Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine. The term “conservative variant” or “conservative variation”also includes the use of a substituting amino acid residue in place ofan amino acid residue in a given parent amino acid sequence, whereantibodies specific for the parent sequence are also specific for, i.e.,“cross-react” or “immuno-react” with, the resulting substitutedpolypeptide sequence.

[0050] “Defect” or “defect site” refers to a site requiring bone, joint,cartilage or ligament repair, construction, fusion, regeneration oraugmentation. The site may be an orthopedic structural disruption orabnormality, or a site where bone does not normally grow. The defectfurther can define an osteochondral defect, including a structuraldisruption of both the bone and overlying cartilage. A defect can assumethe configuration of a “void”, which is understood to mean athree-dimensional defect such as, for example, a gap, cavity, hole orother substantial disruption in the structural integrity of a bone orjoint. A defect can be the result of accident, disease, surgicalmanipulation, and/or prosthetic failure. In certain embodiments, thedefect is a void having a volume incapable of endogenous or spontaneousrepair. Such defects in long bone are generally twice the diameter ofthe subject bone and are also called “critical size” defects. Forexample, in a canine ulna defect model, the art recognizes such defectsto be approximately 3-4 cm. Generally, critical size defects areapproximately 1.0 cm, and incapable of spontaneous repair. See, forexample, Schmitz et al., Clinical Orthopaedics and Related Research,205, pp. 299-308 (1986); and Vukicevic et al., in Advances in Molecularand Cell Biology, 6, pp. 207-224 (1993)(JAI Press, Inc.). In rabbit andmonkey segmental defect models, the gap is approximately 1.5 cm and 2.0cm, respectively. In other embodiments, the defect is a non-criticalsize segmental defect. Generally, these are capable of spontaneousrepair. In certain other embodiments, the defect is an osteochondraldefect, such as an osteochondral plug. Such a defect traverses theentirety of the overlying cartilage and enters, at least in part, theunderlying bony structure. In contrast, a chondral or subchondral defecttraverses the overlying cartilage, in part or in whole, respectively,but does not involve the underlying bone. Other defects susceptible torepair using the instant invention include, but are not limited to,non-union fractures; bone cavities; tumor resection; fresh fractures(distracted or undistracted); cranial, maxillofacial and facialabnormalities, for example, in facial skeletal reconstruction,specifically, orbital floor reconstruction, augmentation of the alveolarridge or sinus, periodontal defects and tooth extraction socket;cranioplasty, genioplasty, chin augmentation, palate reconstruction, andother large bony reconstructions; vertebroplasty, interbody fusions inthe cervical, thoracic and lumbar spine and posteriolateral fusions inthe thoracic and lumbar spine; in osteomyelitis for bone regeneration;appendicular fusion, ankle fusion, total hip, knee and joint fusions orarthroplasty; correcting tendon and/or ligamentous tissue defects suchas, for example, the anterior, posterior, lateral and medial ligamentsof the knee, the patella and achilles tendons, and the like as well asthose defects resulting from diseases such as cancer, arthritis,including osteoarthritis, and other bone degenerative disorders such asosteochondritis dessicans.

[0051] “Granulating solution” refers to a solution that has a certaindegree of consistency and cohesiveness, and enhances the formation ofgranules.

[0052] “Morphogenic protein” refers to a protein having morphogenicactivity (see below). Preferably a morphogenic protein of this inventioncomprises at least one polypeptide belonging to the BMP protein family.Morphogenic proteins may be capable of inducing progenitor cells toproliferate and/or to initiate differentiation pathways that lead tocartilage, bone, tendon, ligament, neural or other types of tissueformation depending on local environmental cues, and thus morphogenicproteins may behave differently in different surroundings. For example,an osteogenic protein may induce bone tissue at one treatment site andneural tissue at a different treatment site.

[0053] “Morphogenic protein stimulatory factor (MPSF)” refers to afactor that is capable of stimulating the ability of a morphogenicprotein to induce tissue formation from a progenitor cell. The MPSF mayhave a direct or indirect effect on enhancing morphogenic proteininducing activity. For example, the MPSF may increase the bioactivity ofanother MPSF. Agents that increase MPSF bioactivity include, forexample, those that increase the synthesis, half-life, reactivity withother biomolecules such as binding proteins and receptors, or thebioavailability of the MPSF.

[0054] “Osteogenic protein (OP)” refers to a morphogenic protein that iscapable of inducing a progenitor cell to form cartilage and/or bone. Thebone may be intramembranous bone or endochondral bone. Most osteogenicproteins are members of the BMP protein family and are thus also BMPs.As described elsewhere herein, the class of proteins is typified byhuman osteogenic protein (hOP-1). Other osteogenic proteins useful inthe practice of the invention include osteogenically active forms ofOP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-9, DPP, Vgl,Vgr, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP-10, BMP-11,BMP-13, BMP-15, UNIVIN, NODAL, SCREW, ADMP or NEURAL and amino acidsequence variants thereof. In one currently preferred embodiment,osteogenic protein includes any one of: OP-1, OP-2, OP-3, BMP-2, BMP-4,BMP-5, BMP-6, BMP-9, and amino acid sequence variants and homologsthereof, including species homologs thereof. Particularly preferredosteogenic proteins are those comprising an amino acid sequence havingat least 70% homology with the C-terminal 102-106 amino acids, definingthe conserved seven cysteine domain, of human OP-1, BMP-2, and relatedproteins. Certain preferred embodiments of the instant inventioncomprise the osteogenic protein, OP-1. As further described elsewhereherein, the osteogenic proteins suitable for use with applicants'invention can be identified by means of routine experimentation usingthe art-recognized bioassay described by Reddi and Sampath (Sampath etal., Proc. Natl. Acad. Sci., 84, pp. 7109-13, incorporated herein byreference)

[0055] Proteins useful in this invention include eukaryotic proteinsidentified as osteogenic proteins (see U.S. Pat. No. 5,011,691,incorporated herein by reference), such as the OP-1, OP-2, OP-3 andCBMP-2 proteins, as well as amino acid sequence-related proteins, suchas DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse), GDF-1(from humans, see Lee, PNAS, 88, pp. 4250-4254 (1991)), 60A (fromDrosophila, see Wharton et al. PNAS, 88, pp. 9214-9218 (1991)),dorsalin-1 (from chick, see Basler et al. Cell 73, pp. 687-702 (1993)and GenBank accession number L12032) and GDF-5 (from mouse, see Storm etal. Nature, 368, pp. 639-643 (1994)). The teachings of the abovereferences are incorporated herein by reference. BMP-3 is alsopreferred. Additional useful proteins include biosynthetic morphogenicconstructs disclosed in U.S. Pat. No. 5,011,691, incorporated herein byreference, e.g., COP-1, COP-3, COP-4, COP-5, COP-7 and COP-16, as wellas other proteins known in the art. Still other proteins includeosteogenically active forms of BMP-3b (see Takao, et al. Biochem.Biophys. Res. Comm., 219, pp. 656-662 (1996)). BMP-9 (see WO95/33830),BMP-15 (see WO96/35710), BMP-12 (see WO95/16035), CDMP-1 (see WO94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893), GDF-1 (seeWO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965) and GDF-7(see WO95/01802). The teachings of the above references are incorporatedherein by reference.

[0056] “Repair” is intended to mean new bone and/or cartilage formationwhich is sufficient to at least partially fill the void or structuraldiscontinuity at the defect. Repair does not, however, mean, orotherwise necessitate, a process of complete healing or a treatmentwhich is 100% effective at restoring a defect to its pre-defectphysiological/structural/mechanical state.

[0057] “Synergistic interaction” refers to an interaction in which thecombined effect of two or more agents is greater than the algebraic sumof their individual effects.

[0058] Porous β-TCP

[0059] This present invention provides a porous β-TCP having a pore sizeand granule size appropriate for bone formation, bone regeneration, andbone repair at a defect site in a human or animal. The porous β-TCP bodydescribed in this invention comprises β-TCP having a multiplicity ofpores. Each pore is a single separate void partitioned by walls and isnot interconnected. The porous β-TCP body of this invention is distinctfrom the cancellous or fenestrate structures that contain capillary voidpaths or interconnections between adjacent pores. The pore diameter sizeof the porous β-TCP of this invention is in the range of 20-500 μm. Inone embodiment, the pore diameter size is in the range of 410-460 um. Ina preferred embodiment, the pore diameter size is 40-190 μm. In anotherembodiment, the pore diameter size is in the range of 20-95 um. In amore preferred embodiment, the pore diameter is in the range of 50-125μm. These pores provide residence spaces for the infiltrating osteolyticcells and osteoblasts when the β-TCP material is embedded in the livingbody. In one embodiment, the pores are spherical and uniformlydistributed. Spherical pores having a diameter in the range of 20-500 μmare appropriate for osteoblast infiltration. Spherical pores alsoprovide the porous body with the necessary mechanical strength duringthe period that new bone is being synthesized, thus preventing the bonefrom fracturing during this period.

[0060] Tricalcium phosphate (TCP) has the formula of Ca₃ (PO₄)₂, withthe Ca/P ratio being about 1.5. TCP powder has an apatite crystalstructure. Upon sintering, the apatite structure converts to the rhombicβ-TCP structure. At high temperatures, the metastable, α-TCP structurecan also form. α-TCP is known to have excessive solubility, which doesnot permit the rate of resorption to be complementary to the rate ofsubstitution by the hard tissue. In addition, α-TCP is capable ofgenerating harmful inflammatory responses. In a preferred embodiment,the TCP is sintered at high temperatures of 1100-1200° C. Above 1300°C., TCP is converted to the metastable α-TCP. Sintering the TCP reducesits solubility in body fluids, which leads to a corresponding reductionin its chemical activity so that the porous TCP is well tolerated in thebody and acute inflammatory reactions are avoided. Therefore, the porousβ-TCP is preferably sintered. More preferably the β-TCP comprises β-TCPthat is 95-100% pure.

[0061] The porous β-TCP material of the present invention may have anyshape and size. In one embodiment, the porous β-TCP is granular and hasa particle size between 0.1 to 2 mm. In a preferred embodiment, theparticle size is 0.5-1.7 mm. In a more preferred embodiment the particlesize is 1.0-1.7 mm. In a most preferred embodiment, the particle size is0.5-1 mm. β-TCP having a granule size of less than 0.1 mm is notappropriate because it will be readily displaced by flowing body fluids.On the other hand, although bone formation is more obvious in largerparticles, β-TCP having a granule size greater than 2 mm is also notappropriate because too many or excessively large gaps will form betweenthe granules, thus preventing the effective coalescence of the β-TCP tothe newly synthesized bone.

[0062] The porosity of the β-TCP influences the resorption rate. If theporosity is too high, the strength of the granules will be decreased. Ifthe porosity is too low, the rate of resorption will be slow. The totalporosity is measured using the mercury intrusion parameter method orequivalent methods. In one embodiment, the total porosity is in therange of 5-80%. In another embodiment, the total porosity is in therange of 40-80%. In a more preferred embodiment, the total porosity is65-75%. In a most preferred embodiment, the total porosity is 70%.

[0063] The porous β-TCP of this invention may also be combined with oneor more bioactive agents. The bioactive agent may be an agent thatenhances bone growth or a substance that is medically useful orcombinations thereof. It is envisioned that the bioactive agent caninclude but is not limited to bone morphogenic proteins, growth factorssuch as EGF, PDGF, IGF, FGF, TGF-α and TGF-β, cytokines, MPSF, hormones,peptides, lipids, trophic agents and therapeutic compositions includingantibiotics and chemotherapeutic agents, insulin, chemoattractant,chemotactic factors, enzymes, enzyme inhibitors. It is also envisionedthat bioactive agents such as vitamins, cytoskeletal agents, cartilagefragments, allografts, autografts, living cells such as chondrocytes,bone marrow cells, mesenchymal stem cells, tissue transplants,immuno-suppressants may be added to the porous β-TCP.

[0064] In one embodiment, the bioactive agent is a bone morphogenicprotein. In a preferred embodiment, the bone morphogenic protein is OP-1(BMP-7), OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2,BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3,GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121,dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP,NEURAL, and TGF-β. In a more preferred embodiment, the morphogenicprotein is OP-1.

[0065] In another embodiment the morphogenic activity of the bonemorphogenic protein is enhanced by the addition of a MPSF. In apreferred embodiment the MPSF is selected from the group consisting ofinsulin-like growth factor I (IGF-I), estradiol, fibroblast growthfactor (FGF), growth hormone (GH), growth and differentiation factor(GDF), hydrocortisone (HC), insulin, progesterone, parathyroid hormone(PTH), vitamin D, retinoic acid and IL-6. In a preferred embodiment, theMPSF is selected from IGF-1, IL-6, FGF, PTH. In a more preferredembodiment, the MPSF is IGF-1.

[0066] In another embodiment, the bioactive agent is preferably anantimicrobial or antibiotic including but not limited to erythromycin,bacitracin, neomycin, penicillin, polymyxin B, tetracycline, viomycin,chloromycetin and streptomycin, cefazolin, ampicillin, azactam,tobramycin, clindamycin and gentamycin. The concentrations of theantibiotic to be used are well known in the art. Such antibiotics havebeen known and used in connection with bone cement materials. See, forexample, Hoff et al., J. Bone Joint Surg., 63A, pp. 798, (1981); andDueland et al., Clin. Orthop., 169, pp. 264-268, (1982). The teachingsof these two references are incorporated herein by reference.

[0067] In another preferred embodiment, the bioactive agent is a repaircell. In a preferred embodiment, the repair cell is a mammalian cell,more preferably, a human cell of the same type as that of the tissuebeing repaired or reconstructed. Suitable examples of repair cellsinclude bone cells such as bone marrow stem cells, osteocytes,osteoblasts, osteoclasts and bone progenitor cells. In anotherembodiment, the cell is transfected with a nucleic acid moleculeencoding a BMP.

[0068] In yet another preferred embodiment, the bioactive agent is anucleic acid molecule comprising a sequence encoding a BMP, preferably,OP-1 (SEQ ID NO: 10). In a preferred embodiment, the nucleic acidmolecule is a RNA or DNA molecule. The nucleic acid sequence encodingthe BMP may be inserted in recombinant expression vectors. Examples ofvectors include but are not limited to pBR322, pH 717, pH 731, pH 752,pH 754 and pW24. SP6 vectors may be used for in vitro transcription ofRNA. Transcription promoters useful for expressing the BMP include butare not limited to the SV40 early promoter, the adenovirus promoter(AdMLP), the mouse metallothionein-I promoter (mMT-I), the Rous sarcomavirus (RSV) long terminal repeat (LTR), the mouse mammary tumor viruslong terminal repeat (MMTV-LTR), and the human cytomegalovirus majorintermediate-early promoter (hCMV). The DNA sequences for all of thesepromoters are known in the art and are available commercially. The DNAsequence may also be inserted in the genome of a recombinant virus suchas, for example recombinant adenovirus, adeno-associated virus orretrovirus. The repair cell or bone progenitor cell is then transfectedor infected with the vector or virus and expresses the BMP protein. Thenucleic acid sequence may transiently or stably transfect the repaircell or bone progenitor cell.

[0069] In one embodiment, the nucleic acid molecule is directly injectedinto the implant site. Preferably, the nucleic acid is trapped in acarrier selected from the group consisting of mannitol, sucrose,lactose, trehalose, liposomes, proteoliposomes that contain viralenvelope proteins and polylysine-glycoprotein complexes. See, e.g.,Ledley, J. Pediatrics 110, pp. 1 (1987); Nicolau et al., Proc. Natl.Acad. Sci. U.S.A., 80, pp. 1068 (1983). In another preferred embodiment,the nucleic acid is transfected or infected into target cells such asbone progenitor cells and repair cells that have been removed from thebody. The transfected cell or infected cells are then re-implanted intothe body.

[0070] In a most preferred embodiment, the bioactive agent isencapsulated in a biodegradable agent. As the biodegradable agent isslowly resorbed by the osteoclast cells, the encapsulated bioactiveagent is gradually released into the matrix. At the implant site, onemay deliver the bioactive agent through a combination of differentbiodegradable agents, preferably, differing in the rate of resorption,to achieve a multiple boost delivery system. In another preferredembodiment, the biodegradable agent is multi-layered. Each layercomprises a different biodegradable agent, preferably, differing in therate of resorption. Methods of encapsulating the bioactive agent includebut are not limited to the emulsion-solvent evaporation method(Grandfils et al., Journal of Biomedical Materials Research, 26, pp.467-479 (1992)) and the method described in Herbert et al.,Pharmaceutical Research, 15, pp. 357-361 (1998). The above referencesare incorporated herein by reference. The latter method is especiallysuitable for encapsulating proteins. Other methods are described in U.S.Pat. Nos. 6,110,503, 5,654,008 and 5,271,961, which are incorporatedherein by reference. In a preferred embodiment, the OP-1 is stabilizedby the addition of lactose during the encapsulation process.

[0071] The biodegradable agents of this invention may be in bead ormicrosphere form. The biodegradable agents can be resorbablebiocompatible polymers including both natural and synthetic polymers.Natural polymers are typically absorbed by enzymatic degradation in thebody, while synthetic resorbable polymers typically degrade by ahydrolytic mechanism. It is preferred that the particle size of thebiodegradable agent is 20-500 μm, preferably, 20-140 μm, more preferably50-140 μm, and most preferably 75-140 μm.

[0072] In one embodiment, the biodegradable agent is selected from thegroup consisting of ethylenevinylacetate, natural and syntheticcollagen, poly(glaxanone), poly(phosphazenes), polyglactin, polyglacticacid, polyaldonic acid, polyacrylic acids, polyalkanoates, ,polyorthoesters, poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA),polyglycolide (PGA), poly(lactide-co-glycolide (PLGA),poly(ζ-caprolactone), poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate) poly(D,L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more α-hydroxy carboxylic acid monomers, bioactiveglass compositions, admixtures thereof and any derivatives andmodifications thereof. Preferably, the modification changes less than50% of the overall structure of the polymer.

[0073] In a preferred embodiment, the biodegradable agent is selectedfrom the group consisting of polyorthoesters, poly(L-lactide) (PLLA),poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(ζ-caprolactone),poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate), poly(D,L-lactide-co-trimethylene carbonate), polyarylatesand co-polymers thereof.

[0074] In another more preferred embodiment, the biodegradable agent isselected from the group consisting of poly(glaxanone),poly(phosphazenes), ethylenevinylacetate, polyglactin, polyglactic acid,polyaldonic acid, polyacrylic acids, polyalkanoates, co-polymers thereofand natural and synthetic collagen.

[0075] In yet another more preferred embodiment the biodegradable agentis selected from the group consisting of polyhydroxybutyrate (PHB),anhydrides including polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more -hydroxy carboxylic acid monomers, (e.g.α-hydroxy acetic acid (glycolic acid) and/or α-hydroxy propionic acid(lactic acid)), bioactive glass compositions. α-hydroxy propionic acidcan be employed in its d- or 1-form, or as a racemic mixture.

[0076] In a most preferred embodiment the biodegradable agent ispoly(lactide-co-glycolide) (PLGA). Depending upon the desired rate ofrelease of the bioactive agent, the molar ratio of the lactide,glycolide monomers can be adjusted. In a preferred embodiment, themonomer ratio is 50:50. In general, the higher the molecular weight, theslower the biodegradation. Preferably, the molecular weight range of thepolymer is from about 5,000 to 500,000 daltons, more preferably 10,000to 30,000 daltons.

[0077] Method of Producing Porous β-TCP

[0078] The invention also relates to a method of producing porous β-TCPgranules. The TCP used in preparing the porous β-TCP is preparedaccording to known methods in the art. The TCP is harvested via a spraydryer, preferably to a particle size of less than 10 μm. If the particlesize is too large, it will interfere with the formation of pores.

[0079] The fine TCP powder is then mixed with a pore-forming agent thatdecomposes at high temperature into gaseous decomposition productswithout leaving any solid residue. The pore-forming agents of thisinvention may be in bead or resin form. In one embodiment, thepore-forming agents are selected from thermally decomposable materialsuch as naphthalene, prepolymers of polyacrylates, prepolymers ofpolymethacrylates, polymethyl methacrylate, copolymers of methylacrylate and methyl methacrylate and mixtures thereof, polystyrene,polyethylene glycol, crystalline cellulose powder, fibrous cellulose,polyurethanes, polyethylenes, nylon resins and acrylic resins. In a morepreferred embodiment the pore-forming agent is selected from the groupconsisting of polymethyl methacrylate, polystyrene and polyethyleneglycol. It is preferred that the pore-forming agent creates a pore sizediameter of 20-500 μm, more preferably 40-190 μm, and most preferably50-125 μm after sintering.

[0080] The proportion and particle size of the pore-forming agentinfluences the porosity and the pore structure. An excessive amount ofthe pore-forming agent leads to interconnected pores and a decrease indensity of the β-TCP body and hence mechanical strength of the sinteredbody. A deficiency in the amount of the pore-forming agent may result inthe insufficiently developed pore structure. The proportion ofpore-forming agent is preferably 10-50% by weight, more preferably30-40% by weight, most preferably 37.5% by weight.

[0081] A granulating solution is then added to the mixture of TCP powderand pore-forming agent to produce a crumbly mass. This improves thesieving procedure that follows. Depending on the desired viscosity to beachieved and the aqueous properties of the dispersing medium, thecompound used to form the granulating solution may be selected from thegroup consisting of polyvinyl pyrrolidone, starch, gelatin, polyvinylalcohol, polyethylene oxide, hydroxyethyl cellulose, polyvinyl butyraland cellulose acetate butyrate. Preferably, the compound in thegranulating solution is selected from the group consisting of polyvinylpyrrolidone, starch and gelatin.

[0082] The crumbly mass is then sieved to select for a range of granulesizes. The size of the granules selected by the sieving process may bein the range of 250-1700 μm, more preferably 1000-1700 μm, mostpreferably 500-1000 μm. The sieved granules are then dried at 90-110°C., more preferably at 105° C.

[0083] The dried granules are then heated to 700-800° C. to remove thepore-forming agent. The temperature is then raised to 1000-1200° C.,more preferably 1150° C., for sintering. The sintered granules undergo aslow cooling procedure to attain pure crystalline β-TCP. In a preferredembodiment the temperature is lowered from 1150° C. to 39° C. in 6hours. After sintering, weight loss and shrinkage takes place in thesample. Pores are formed in the TCP and the pores are surrounded by theskeleton of sintered TCP. The sintered granules are resieved using thesame size sieve as previously used and mixed with a binder as previouslydescribed to form a moldable putty composition.

[0084] Alternatively, the porous β-TCP granules may be prepared bymixing the TCP powder with the pore-forming agent. The mixture isblended to achieve homogeneity and pressed into slugs using a press,rotary tablet machine or chilsonators. The slugs are heated to 700-800°C. to remove the pore-forming agent and sintered at 1000-1100° C.,preferably at 1150° C. The porous slugs are then fractured into theappropriate particle size range of 250-1700 μm, more preferably1000-1700 μm, and most preferably 500-1000 μm. The porous granules arethen mixed with a binder to form a moldable putty composition.

[0085] Moldable Putty Composition

[0086] The porous β-TCP of this invention may be combined with abiocompatible binder to form a moldable putty composition. The moldableputty may be in the form of a paste or a semi-solid having sufficientviscosity. The moldable putty composition enables the positioning andshaping within the voids, defects or other areas in which new bonegrowth is desired. The cohesiveness of the putty also prevents theproblems of particle migration associated with grafting materials fororthopedic, maxillofacial and dental applications.

[0087] The binder according to this invention must be biodegradable,biocompatible and have fluid flow properties. The binders contemplatedas useful herein include, but are not limited to: art-recognizedsuspending agents, viscosity-producing agents, gel-forming agents andemulsifying agents. Other candidates are agents used to suspendingredients for topical, oral or parental administration. Yet othercandidates are agents useful as tablet binders, disintegrants oremulsion stabilizers. Still other candidates are agents used incosmetics, toiletries and food products. Reference manuals such as theUSP XXII -NF XVII (The Nineteen Ninety U.S. Pharmacopeia and theNational Formulary (1990)) categorize and describe such agents.Preferred binders include resorbable macromolecules from biological orsynthetic sources including sodium alginate, hyaluronic acid, cellulosederivatives such as alkylcelluloses including methylcellulose, carboxymethylcellulose, carboxy methylcellulose sodium, carboxy methylcellulosecalcium or other salts, hydroxy alkylcelluloses including hydroxypropylmethylcellulose, hydroxybutyl methylcellulose, hydroxyethylmethylcellulose, hydroxyethyl cellulose, alkylhydroxyalkyl cellulosesincluding methylhydroxyethyl cellulose, collagen, peptides, mucin,chrondroitin sulfate and the like.

[0088] Carboxymethylcellulose (CMC) sodium is a preferred binder. CMC iscommercially available from suppliers such as, but not limited to:Hercules Inc., Aqualon® Division, Delaware; FMC Corporation,Pennsylvania; British Celanese, Ltd., United Kingdom; and Henkel KGaA,United Kingdom. Carboxymethylcellulose sodium is the sodium salt of apolycarboxymethyl ether of cellulose with a typical molecular weightranging from 90,000-700,000. Various grades of carboxymethylcellulosesodium are commercially available which have differing viscosities.Viscosities of various grades of carboxymethylcellulose sodium arereported in Handbook of Pharmaceutical Excipients (2nd Edition),American Pharmaceutical Association & Royal Pharmaceutical Society ofGreat Britain. For example, low viscosity 50-200 cP, medium viscosity400-800 cP, high viscosity 1500-3000 cP. A number of grades ofcarboxymethylcellulose sodium are commercially available, the mostfrequently used grade having a degree of substitution (DS) of 0.7. TheDS is defined as the average number of hydroxyl groups substituted peranhydroglucose unit. It is this DS which determines the aqueoussolubility of the polymer. The degree of substitution and the standardviscosity of an aqueous solution of stated concentration is indicated onany carboxymethylcellulose sodium labeling. Low viscosity CMC (Aqualon®Division, Hercules, Inc., Wilmington, Del.) is currently preferred. Thecurrently preferred degrees of substitution range from 0.65-0.90(DS=0.7, Aqualon® Type 7L).

[0089] Aside from binders that are flowable at room temperature, bindersalso include reagents such as gelatin, that are solubilized in warm orhot aqueous solutions, and are transformed into a non-flowable gel uponcooling. The gelatin composition is formulated so that the compositionis flowable at temperatures above the body temperature of the mammal forimplant, but transitions to relatively non-flowable gel at or slightlyabove such body temperature.

[0090] In one embodiment, the binder of this invention is selected froma class of high molecular weight hydrogels including sodium hyaluronate(˜500-3000 kD), chitosan (˜100-300 kD), poloxamer (˜7-18 kD), andglycosaminoglycan (˜2000-3000 kD). In a preferred embodiment, theglycosaminoglycan is N,O-carboxymethylchitosan glucosamine. Hydrogelsare cross-linked hydrophilic polymers in the form of a gel which have athree-dimensional network. Hydrogel matrices can carry a net positive ornet negative charge, or may be neutral. A typical net negative chargedmatrix is alginate. Hydrogels carrying a net positive charge may betypified by extracellular matrix components such as collagen andlaminin. Examples of commercially available extracellular matrixcomponents include Matrigel™ (Dulbecco's modified eagle's medium with 50μg/ml gentamicin) and Vitrogen™ (a sterile solution of purified,pepsin-solubilized bovine dermal collagen dissolved in 0.012 N HCL). Anexample of a net neutral hydrogel is highly crosslinked polyethyleneoxide, or polyvinyalcohol.

[0091] In another embodiment, the binder of this invention may also beselected from a class of polymers selected from the group comprisingpolylactic acid, polyglycolic acid, co-polymers of polylactic acid andpolyglycolic acid, polyhydroxybutyric acid, polymalic acid, polyglutamicacid, and polylactone. In order to have gradual polymer replacement inthe material by in situ tissue ingrowth over a several-day toseveral-week period, the molecular weight of the polymer should becompatible with the required degradation rate of the polymer.

[0092] In another preferred embodiment, the binder is polyethyleneglycol. A mixture of low- and high-molecular-weight polyethylene glycolscan produce a paste with the proper viscosity. For example, a mixture ofpolyethylene glycols of molecular weight 400-600 daltons and 1500daltons at the proper ratio would be effective.

[0093] In yet another embodiment, the binder is selected from a class ofpolysaccharides with an average molecular weight of about 200,000 to5,000,000 daltons consisting of dextran, dextran sulfate,diethylaminoethyl dextran, dextran phosphate or mixtures thereof. Lowermolecular weight polysaccharides have the advantage of a faster dextranabsorption rate, resulting in earlier exposure of the porous β-TCPmaterial. If it is desired that dextrans remain in the site for anextended period, dextrans of relatively high molecular weight may beused. Other preferred polysaccharides include starch, fractionatedstarch, amylopectin, agar, gum arabic, pullullan, agarose, carrageenan,dextrins, fructans, inulin, mannans, xylans, arabinans, glycogens,glucans, xanthan gum, guar gum, locust bean gum, tragacanth gum, karayagum, and derivatives and mixtures thereof.

[0094] In another preferred embodiment, the binder is selected from thegroup consisting of mannitol, white petrolatum, mannitol/dextrancombinations, mannitol/white petrolatum combinations, sesame oil, fibringlue and admixtures thereof. Fibrin glue is currently a preferredbinder, which comprises a mixture of mammalian fibrinogen and thrombin.Human fibrinogen is commercially available in products such as, but notlimited to Tissucol® (Immuno AG, Vienna, Austria), Beriplast®(Behringwerke, Marburg, Germany), Biocoll® (Centre de TransfusionSanguine de Lille, Pours, France) and Transglutine® (CNTS FractionationCentre, Strasbourg, France). Fibrin glue may also be made of fibrinogenand thrombin from other mammalian sources, such as, for example, bovineand murine sources.

[0095] It is preferred that the binder is selected from the groupconsisting of sodium alginate, hyaluronic acid, sodium hyaluronate,gelatin, collagen, peptides, mucin, chrondroitin sulfate, chitosan,poloxamer, glycosaminoglycan, polysaccharide, polyethylene glycol,methylcellulose, carboxy methylcellulose, carboxy methylcellulosesodium, carboxy methylcellulose calcium, hydroxypropyl methylcellulose,hydroxybutyl methylcellulose, hydroxyethyl methylcellulose,hydroxyethylcellulose, methylhydroxyethyl cellulose, hydroxyethylcellulose, polylactic acid, polyglycolic acid, co-polymers of polylacticacid and polyglycolic acid, polyhydroxybutyric acid, polymalic acid,polyglutamic acid, polylactone, mannitol, white petrolatum,mannitol/dextran combinations, mannitol/white petrolatum combinations,sesame oil, fibrin glue and admixtures thereof.

[0096] More preferably, the binder is selected from the group consistingof sodium alginate, hyaluronic acid, methylcellulose, carboxymethylcellulose, carboxy methylcellulose sodium, carboxy methylcellulosecalcium, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose,hydroxyethyl methylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxyethyl cellulose and admixtures thereof. Mostpreferably, the binder is selected from the group consisting of sodiumalginate, hyaluronic acid, carboxy methylcellulose, carboxymethylcellulose sodium and carboxy methylcellulose calcium.

[0097] The minimum amount of binder is the amount necessary to give easyformability and provide sufficient particle cohesion and shape retentionduring the period of tissue ingrowth. In one embodiment, the weightratio of porous β-TCP to carboxy methylcellulose sodium is in the rangeof 1:0.1 to 1:1.25. In a preferred embodiment, the ratio of porous β-TCPto CMC sodium is 1:0.4.

[0098] The invention also relates to a kit for bone implant comprisingthe porous β-TCP material of the invention and at least one additionalbioactive agent selected from the group consisting of bone morphogenicproteins and antibiotics. The kit comprising the porous β-TCP materialand a bone morphogenic protein may further comprise a morphogenicprotein stimulatory factor. In one embodiment, the kit further comprisesa binder. In another embodiment, the kit comprises the porous β-TCPmaterial of the invention and a binder.

[0099] Bone Morphogenic Protein Family

[0100] The BMP family, named for its representative bonemorphogenic/osteogenic protein family members, belongs to the TGF-βprotein superfamily. Of the reported “BMPs” (BMP-1 to BMP-18), isolatedprimarily based on sequence homology, all but BMP-1 remain classified asmembers of the BMP family of morphogenic proteins (Ozkaynak et al., EMBOJ., 9, pp. 2085-93 (1990)).

[0101] The BMP family includes other structurally-related members whichare morphogenic proteins, including the drosophila decapentaplegic genecomplex (DPP) products, the Vgl product of Xenopus laevis and its murinehomolog, Vgr-1 (see, e.g., Massagué, Annu. Rev. Cell Biol., 6, pp.597-641 (1990), incorporated herein by reference).

[0102] The C-terminal domains of BMP-3, BMP-5, BMP-6, and OP-1 (BMP-7)are about 60% identical to that of BMP-2, and the C-terminal domains ofBMP-6 and OP-1 are 87% identical. BMP-6 is likely the human homolog ofthe murine Vgr-1 (Lyons et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.4554-59 (1989)); the two proteins are 92% identical overall at the aminoacid sequence level (U.S. Pat. No. 5,459,047, incorporated herein byreference). BMP-6 is 58% identical to the Xenopus Vg-1 product.

[0103] Biochemical Structural and Functional Properties of BoneMorphogenic Proteins

[0104] The naturally occurring bone morphogens share substantial aminoacid sequence homology in their C-terminal regions (domains). Typically,the above-mentioned naturally occurring osteogenic proteins aretranslated as a precursor, having an N-terminal signal peptide sequencetypically less than about 30 residues, followed by a “pro” domain thatis cleaved to yield the mature C-terminal domain of approximately100-140 amino acids. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne Nucleic Acids Research, 14, pp.4683-4691 (1986). The pro domain typically is about three times largerthan the fully processed mature C-terminal domain.

[0105] Another characteristic of the BMP protein family members is theirapparent ability to dimerize. Several bone-derived osteogenic proteins(OPs) and BMPs are found as homo- and heterodimers in their activeforms. The ability of OPs and BMPs to form heterodimers may conferadditional or altered morphogenic inductive capabilities on morphogenicproteins. Heterodimers may exhibit qualitatively or quantitativelydifferent binding affinities than homodimers for OP and BMP receptormolecules. Altered binding affinities may in turn lead to differentialactivation of receptors that mediate different signaling pathways, whichmay ultimately lead to different biological activities or outcomes.Altered binding affinities could also be manifested in a tissue or celltype-specific manner, thereby inducing only particular progenitor celltypes to undergo proliferation and/or differentiation.

[0106] In preferred embodiments, the pair of morphogenic polypeptideshave amino acid sequences each comprising a sequence that shares adefined relationship with an amino acid sequence of a referencemorphogen. Herein, preferred osteogenic polypeptides share a definedrelationship with a sequence present in osteogenically active humanOP-1, SEQ ID NO: 1. However, any one or more of the naturally occurringor biosynthetic sequences disclosed herein similarly could be used as areference sequence. Preferred osteogenic polypeptides share a definedrelationship with at least the C-terminal six cysteine domain of humanOP-1, residues 335-431 of SEQ ID NO: 1. Preferably, osteogenicpolypeptides share a defined relationship with at least the C-terminalseven cysteine domain of human OP-1, residues 330-431 of SEQ ID NO: 1.That is, preferred polypeptides in a dimeric protein with bonemorphogenic activity each comprise a sequence that corresponds to areference sequence or is functionally equivalent thereto.

[0107] Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy bone morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al., supra, the teachings ofwhich are incorporated by reference herein.

[0108] Conservative substitutions typically include the substitution ofone amino acid for another with similar characteristics, e.g.,substitutions within the following groups: valine, glycine; glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. The term “conservative variation” also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid provided that antibodies raised to the substitutedpolypeptide also immunoreact with the unsubstituted polypeptide.

[0109] The osteogenic protein OP-1 has been described (see, e.g.,Oppermann et al., U.S. Pat. No. 5,354,557, incorporated herein byreference). Natural-sourced osteogenic protein in its mature, nativeform is a glycosylated dimer typically having an apparent molecularweight of about 30-36 kDa as determined by SDS-PAGE. When reduced, the30 kDa protein gives rise to two glycosylated peptide subunits havingapparent molecular weights of about 16 kDa and 18 kDa. In the reducedstate, the protein has no detectable osteogenic activity. Theunglycosylated protein, which also has osteogenic activity, has anapparent molecular weight of about 27 kDa. When reduced, the 27 kDaprotein gives rise to two unglycosylated polypeptides, having molecularweights of about 14 kDa to 16 kDa, capable of inducing endochondral boneformation in a mammal. Osteogenic proteins may include forms havingvarying glycosylation patterns, varying N-termini, and active truncatedor mutated forms of native protein. As described above, particularlyuseful sequences include those comprising the C-terminal 96 or 102 aminoacid sequences of DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (frommouse), the OP-1 and OP-2 proteins,(see U.S. Pat. No. 5,011,691 andOppermann et al., incorporated herein by reference), as well as theproteins referred to as BMP-2, BMP-3, BMP-4 (see WO88/00205, U.S. Pat.No. 5,013,649 and WO91/18098, incorporated herein by reference), BMP-5and BMP-6 (see WO90/11366, PCT/US90/01630, incorporated herein byreference), BMP-8 and BMP-9.

[0110] Preferred morphogenic and osteogenic proteins of this inventioncomprise at least one polypeptide selected from the group consisting ofOP-1 (BMP-7), OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16,BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11,BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2,GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121,dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP,NEURAL, TGF-β and amino acid sequence variants and homologs thereof,including species homologs, thereof. Preferably, the morphogenic proteincomprises at least one polypeptide selected from the group consisting ofOP-1 (BMP-7), BMP-2, BMP-4, BMP-5 and BMP-6; more preferably, OP-1(BMP-7)and BMP-2; and most preferably, OP-1 (BMP-7).

[0111] Publications disclosing these sequences, as well as theirchemical and physical properties, include: OP-1 and OP-2 (U.S. Pat. No.5,011,691; U.S. Pat. No. 5,266,683; Ozkaynak et al., EMBO J., 9, pp.2085-2093 (1990); OP-3 (WO94/10203 (PCT US93/10520)), BMP-2, BMP-3,BMP-4, (WO88/00205; Wozney et al. Science, 242, pp. 1528-1534 (1988)),BMP-5 and BMP-6, (Celeste et al., PNAS, 87, 9843-9847 (1991)), Vgr-1(Lyons et al., PNAS, 86, pp. 4554-4558 (1989)); DPP (Padgett et al.Nature, 325, pp. 81-84 (1987)); Vg-1 (Weeks, Cell, 51, pp. 861-867(1987)); BMP-9 (WO95/33830 (PCT/US95/07084); BMP-10 (WO94/26893(PCT/US94/05290); BMP-11 (WO94/26892 (PCT/US94/05288); BMP-12(WO95/16035 (PCT/US94/14030); BMP-13 (WO95/16035 (PCT/US94/14030); GDF-1(WO92/00382 (PCT/US91/04096) and Lee et al. PNAS, 88, pp. 4250-4254(1991); GDF-8 (WO94/21681 (PCT/US94/03019); GDF-9 (WO94/15966(PCT/US94/00685); GDF-10 (WO95/10539 (PCT/US94/11440); GDF-11(WO96/01845 (PCT/US95/08543); BMP-15 (WO96/36710 (PCT/US96/06540);MP-121 (WO96/01316 (PCT/EP95/02552); GDF-5 (CDMP-1, MP52) (WO94/15949(PCT/US94/00657) and WO96/14335 (PCT/US94/12814) and WO93/16099(PCT/EP93/00350)); GDF-6 (CDMP-2, BMP13) (WO95/01801 (PCT/US94/07762)and WO96/14335 and WO95/10635 (PCT/US94/14030)); GDF-7 (CDMP-3, BMP12)(WO95/10802 (PCT/US94/07799) and WO95/10635 (PCT/US94/14030)) The abovepublications are incorporated herein by reference. In anotherembodiment, useful proteins include biologically active biosyntheticconstructs, including novel biosynthetic morphogenic proteins andchimeric proteins designed using sequences from two or more knownmorphogens.

[0112] In another embodiment of this invention, a morphogenic proteinmay be prepared synthetically for use in concert with a MPSF to inducetissue formation. Morphogenic proteins prepared synthetically may benative, or may be non-native proteins, i.e., those not otherwise foundin nature.

[0113] Non-native osteogenic proteins have been synthesized using aseries of consensus DNA sequences (U.S. Pat. No. 5,324,819, incorporatedherein by reference). These consensus sequences were designed based onpartial amino acid sequence data obtained from natural osteogenicproducts and on their observed homologies with other genes reported inthe literature having a presumed or demonstrated developmental function.

[0114] Several of the biosynthetic consensus sequences (called consensusosteogenic proteins or “COPs”) have been expressed as fusion proteins inprokaryotes. Purified fusion proteins may be cleaved, refolded, combinedwith at least one MPSF (optionally in a matrix or device), implanted inan established animal model and shown to have bone- and/orcartilage-inducing activity. The currently preferred syntheticosteogenic proteins comprise two synthetic amino acid sequencesdesignated COP-5 (SEQ. ID NO: 2) and COP-7 (SEQ. ID NO: 3)

[0115] Oppermann et al., U.S. Pat. Nos. 5,011,691 and 5,324,819, whichare incorporated herein by reference, describe the amino acid sequencesof COP-5 and COP-7 as shown below: COP5LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD COP7LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD COP5HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP7HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP5 ISMLYLDENEKVVLKYNQEMVVEGCGCRCOP7 ISMLYLDENEKVVLKYNQENVVEGCGCR

[0116] In these amino acid sequences, the dashes (−) are used as fillersonly to line up comparable sequences in related proteins. Differencesbetween the aligned amino acid sequences are highlighted.

[0117] The DNA and amino acid sequences of these and other BMP familymembers are published and may be used by those of skill in the art todetermine whether a newly identified protein belongs to the BMP family.New BMP-related gene products are expected by analogy to possess atleast one morphogenic activity and thus classified as a BMP.

[0118] In one preferred embodiment of this invention, the morphogenicprotein comprises a pair of subunits disulfide bonded to produce adimeric species, wherein at least one of the subunits comprises arecombinant peptide belonging to the BMP protein family. In anotherpreferred embodiment of this invention, the morphogenic proteincomprises a pair of subunits that produce a dimeric species formedthrough non-covalent interactions, wherein at least one of the subunitscomprises a recombinant peptide belonging to the BMP protein family.Non-covalent interactions include Van der Waals, hydrogen bond,hydrophobic and electrostatic interactions. The dimeric species may be ahomodimer or heterodimer and is capable of inducing cell proliferationand/or tissue formation.

[0119] In certain preferred embodiments, bone morphogenic proteinsuseful herein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity, with areference morphogenic protein selected from the foregoing naturallyoccurring proteins. Preferably, the reference protein is human OP-1, andthe reference sequence thereof is the C-terminal seven cysteine domainpresent in osteogenically active forms of human OP-1, residues 330-431of SEQ ID NO: 1. In certain embodiments, a polypeptide suspected ofbeing functionally equivalent to a reference morphogen polypeptide isaligned therewith using the method of Needleman, et al., supra,implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). As noted above, internal gaps and amino acid insertionsin the candidate sequence are ignored for purposes of calculating thedefined relationship, conventionally expressed as a level of amino acidsequence homology or identity, between the candidate and referencesequences. “Amino acid sequence homology” is understood herein toinclude both amino acid sequence identity and similarity. Homologoussequences share identical and/or similar amino acid residues, wheresimilar residues are conservation substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina currently preferred embodiment, the reference sequence is OP-1. Bonemorphogenic proteins useful herein accordingly include allelic,phylogenetic counterpart and other variants of the preferred referencesequence, whether naturally-occurring or biosynthetically produced(e.g., including “muteins” or “mutant proteins”), as well as novelmembers of the general morphogenic family of proteins, including thoseset forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

[0120] In another embodiment, useful osteogenic proteins include thosesharing the conserved seven cysteine domain and sharing at least 70%amino acid sequence homology (similarity) within the C-terminal activedomain, as defined herein. In still another embodiment, the osteogenicproteins of the invention can be defined as osteogenically activeproteins having any one of the generic sequences defined herein,including OPX (SEQ ID NO: 4) and Generic Sequences 7 (SEQ ID NO: 5) and8 (SEQ ID NO: 6), or Generic Sequences 9 (SEQ ID NO: 7) and 10 (SEQ IDNO: 8).

[0121] The family of bone morphogenic polypeptides useful in the presentinvention, and members thereof, can be defined by a generic amino acidsequence. For example, Generic Sequence 7 (SEQ ID NO: 5) and GenericSequence 8 (SEQ ID NO: 6) are 97 and 102 amino acid sequences,respectively, and accommodate the homologies shared among preferredprotein family members identified to date, including at least OP-1,OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1,and GDF-1. The amino acid sequences for these proteins are describedherein and/or in the art, as summarized above. The generic sequencesinclude both the amino acid identity shared by these sequences in theC-terminal domain, defined by the six and seven cysteine skeletons(Generic Sequences 7 and 8, respectively), as well as alternativeresidues for the variable positions within the sequence. The genericsequences provide an appropriate cysteine skeleton where inter- orintramolecular disulfide bonds can form, and contain certain criticalamino acids likely to influence the tertiary structure of the foldedproteins. In addition, the generic sequences allow for an additionalcysteine at position 36 (Generic Sequence 7) or position 41 (GenericSequence 8), thereby encompassing the morphogenically active sequencesof OP-2 and OP-3. Generic Sequence 7             Leu Xaa Xaa Xaa Phe XaaXaa             1               5 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa XaaPro         10                  15 Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys XaaGly         20                  25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa XaaXaa         30                  35 Xaa Xaa Xaa Asn His Ala Xaa Xaa XaaXaa         40                  45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa         5 0                  5 5 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa XaaXaa         60                  65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa XaaXaa         70                  75 Xaa Xaa Xaa Val Xaa Leu Xaa Xaa XaaXaa         80                  85 Xaa Met Xaa Val Xaa Xaa Cys Xaa CysXaa         90                  95

[0122] wherein each Xaa independently is selected from a group of one ormore specified amino acids defined as follows: “res.” means “residue”and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa atres.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaaat res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn orGlu); Xaa at res.13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa atres.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or Ser); Xaa atres.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu orGly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr,Asp, Gln, Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa atres.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu orTyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thror Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly orLeu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly orPro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr);Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res.46=(Gln or Arg); Xaa atres.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Valor Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn,Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu);Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Seror Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa atres.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa atres.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa atres.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

[0123] Generic Sequence 8 (SEQ ID NO: 6) includes all of GenericSequence 7 and in addition includes the following sequence (SEQ ID NO:9) at its N-terminus: Cys Xaa Xaa Xaa Xaa SEQ ID NO: 9 1               5

[0124] Accordingly, beginning with residue 7, each “Xaa” in GenericSequence 8 is a specified amino acid defined as for Generic Sequence 7,with the distinction that each residue number described for GenericSequence 7 is shifted by five in Generic Sequence 8. Thus, “Xaa atres.2=(Tyr or Lys)” in Generic Sequence 7 refers to Xaa at res. 7 inGeneric Sequence 8. In Generic Sequence 8, Xaa at res.2=(Lys, Arg, Alaor Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln);and Xaa at res. 5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

[0125] In another embodiment, useful osteogenic proteins include thosedefined by Generic Sequences 9 and 10, defined as follows.

[0126] Specifically, Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-8, human BMP-9, human BMP 10, human BMP-11, Drosophila 60A, XenopusVg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouseGDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouseGDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, DrosophilaSCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like GenericSequence 7, Generic Sequence 9 is a 97 amino acid sequence thataccommodates the C-terminal six cysteine skeleton and, like GenericSequence 8, Generic Sequence 10 is a 102 amino acid sequence whichaccommodates the seven cysteine skeleton. Generic Sequence 9 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1           5                       10 XaaXaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa            15                      20 Xaa Xaa Xaa Xaa Cys Xaa GlyXaaCys Xaa             25                      30 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa             35                      40 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa             45                      50 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa            55                      60 Xaa Cys Xaa Pro Xaa Xaa Xaa XaaXaa Xaa             65                      70 Xaa Xaa Leu Xaa Xaa XaaXaa Xaa Xaa Xaa             75                      80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa             85                      90 Xaa XaaXaa Cys Xaa Cys Xaa             95

[0127] wherein each Xaa is independently selected from a group of one ormore specified amino acids defined as follows: “res.” means “residue”and Xaa at res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg,Thr, Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa atres. 4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa atres. 6=(Arg, Gln, Lys, Ser, Glu, S Ala or Asn); Xaa at res. 7=(Asp, Glu,Leu, Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gin); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gin or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gin, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T ; after res. 54 in BMP3 there is aV; after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro. Generic Sequence 10 (SEQ ID NO: 8)includes all of Generic Sequence 9 (SEQ ID NO: 7) and in additionincludes the following sequence (SEQ ID NO: 9) at its N-terminus: CysXaa Xaa Xaa Xaa SEQ ID NO: 9 1               5

[0128] Accordingly, beginning with residue 6, each “Xaa” in GenericSequence 10 is a specified amino acid defined as for Generic Sequence 9,with the distinction that each residue number described for GenericSequence 9 is shifted by five in Generic Sequence 10. Thus, “Xaa at res.1=(Tyr, Phe, His, Arg, Thr, Lys, Gin, Val or Glu)” in Generic Sequence 9refers to Xaa at res. 6 in Generic Sequence 10. In Generic Sequence 10,Xaa at res. 2=(Lys, Arg, Gin, Ser, His, Glu, Ala, or Cys); Xaa at res.3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln,Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gin, Thr, His,Arg, Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

[0129] As noted above, certain currently preferred bone morphogenicpolypeptide sequences useful in this invention have greater than 60%identity, preferably greater than 65% identity, with the amino acidsequence defining the preferred reference sequence of hOP-1. Theseparticularly preferred sequences include allelic and phylogeneticcounterpart variants of the OP-1 and OP-2 proteins, including theDrosophila 60A protein. Accordingly, in certain particularly preferredembodiments, useful morphogenic proteins include active proteinscomprising pairs of polypeptide chains within the generic amino acidsequence herein referred to as “OPX” (SEQ ID NO: 4), which defines theseven cysteine skeleton and accommodates the homologies between severalidentified variants of OP-1 and OP-2. As described therein, each Xaa ata given position independently is selected from the residues occurringat the corresponding position in the C-terminal sequence of mouse orhuman OP-1 or OP-2. Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp LeuGly Trp Xaa Asp Trp1               5                   10                  15 Xaa Ile AlaPro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                  25                  30                  35 LeuXaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa        40                  45                  50                  55Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala            60                  65                  70 Xaa Ser Val LeuTyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg75                  80                  85                  90 Asn MetVal Val Xaa Ala Cys Gly Cys His         95                  100

[0130] wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg);Xaa at res. 11=(Arg or Gin); Xaa at res. 16=(Gln or Leu); Xaa at res.19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala orSer); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa atres. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Seror Thr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaaat res. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Ginor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

[0131] In still another preferred embodiment, useful osteogenicallyactive proteins have polypeptide chains with amino acid sequencescomprising a sequence encoded by a nucleic acid that hybridizes, underlow, medium or high stringency hybridization conditions, to DNA or RNAencoding reference morphogen sequences, e.g., C-terminal sequencesdefining the conserved seven cysteine domains of OP-1, OP-2, BMP-2,BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-6, GDF-7 and the like. As usedherein, high stringent hybridization conditions are defined ashybridization according to known techniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C. overnight, and washing in0.1×SSPE, 0.1% SDS at 50° C. Standard stringent conditions are wellcharacterized in commercially available, standard molecular cloningtexts. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed.,ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984): Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984), the disclosures of whichare incorporated herein by reference.

[0132] As noted above, proteins useful in the present inventiongenerally are dimeric proteins comprising a folded pair of the abovepolypeptides. Such morphogenic proteins are inactive when reduced, butare active as oxidized homodimers and when oxidized in combination withothers of this invention to produce heterodimers. Thus, members of afolded pair of morphogenic polypeptides in a morphogenically activeprotein can be selected independently from any of the specificpolypeptides mentioned above.

[0133] The bone morphogenic proteins useful in the materials and methodsof this invention include proteins comprising any of the polypeptidechains described above, whether isolated from naturally-occurringsources, or produced by recombinant DNA or other synthetic techniques,and includes allelic and phylogenetic counterpart variants of theseproteins, as well as muteins thereof, and various truncated and fusionconstructs. Deletion or addition mutants also are envisioned to beactive, including those which may alter the conserved C-terminal six orseven cysteine domain, provided that the alteration does notfunctionally disrupt the relationship of these cysteines in the foldedstructure. Accordingly, such active forms are considered the equivalentof the specifically described constructs disclosed herein. The proteinsmay include forms having varying glycosylation patterns, varyingN-termini, a family of related proteins having regions of amino acidsequence homology, and active truncated or mutated forms of native orbiosynthetic proteins, produced by expression of recombinant DNA in hostcells.

[0134] The bone morphogenic proteins contemplated herein can beexpressed from intact or truncated cDNA or from synthetic DNAs inprokaryotic or eukaryotic host cells, and purified, cleaved, refolded,and dimerized to form morphogenically active compositions. Currentlypreferred host cells include, without limitation, prokaryotes includingE. coli or eukaryotes including yeast, or mammalian cells, such as CHO,COS or BSC cells. One of ordinary skill in the art will appreciate thatother host cells can be used to advantage. Detailed descriptions of thebone morphogenic proteins useful in the practice of this invention,including how to make, use and test them for osteogenic activity, aredisclosed in numerous publications, including U.S. Pat. Nos. 5,266,683and 5,011,691, the disclosures of which are incorporated by referenceherein, as well as in any of the publications recited herein, thedisclosures of which are incorporated herein by reference.

[0135] Thus, in view of this disclosure and the knowledge available inthe art, skilled genetic engineers can isolate genes from cDNA orgenomic libraries of various different biological species, which encodeappropriate amino acid sequences, or construct DNAs fromoligonucleotides, and then can express them in various types of hostcells, including both prokaryotes and eukaryotes, to produce largequantities of active proteins capable of stimulating endochondral bonemorphogenesis in a mammal.

[0136] Morphogenic Protein Stimulatory Factors (MPSF)

[0137] A morphogenic protein stimulatory factor (MPSF) according to thisinvention is a factor that is capable of stimulating the ability of amorphogenic protein to induce tissue formation from a progenitor cell.The MPSF may have an additive effect on tissue induction by themorphogenic protein. Preferably, the MPSF has a synergistic effect ontissue induction by the morphogenic protein.

[0138] The progenitor cell that is induced to proliferate and/ordifferentiate by the morphogenic protein of this invention is preferablya mammalian cell. Progenitor cells include mammalian chondroblasts,myoblasts, osteoblasts, neuroblasts and vascular tissue precursor cells,all earlier developmental precursors thereof, and all cells that developtherefrom (e.g., chondroblasts, pre-chondroblasts and chondrocytes).However, morphogenic proteins are highly conserved throughout evolution,and non-mammalian progenitor cells are also likely to be stimulated bysame- or cross-species morphogenic proteins and MPSF combinations. It isthus envisioned that when schemes become available for implantingxenogeneic cells into humans without causing adverse immunologicalreactions, non-mammalian progenitor cells stimulated by morphogenicprotein and a MPSF according to the procedures set forth herein will beuseful for tissue regeneration and repair in humans.

[0139] One or more MPSFs are selected for use in concert with one ormore morphogenic proteins according to the desired tissue type to beinduced and the site at which the morphogenic protein and MPSF will beadministered. The particular choice of a morphogenic protein(s)/MPSF(s)combination and the relative concentrations at which they are combinedmay be varied systematically to optimize the tissue type induced at aselected treatment site using the procedures described herein.

[0140] The preferred morphogenic protein stimulatory factors (MPSFS) ofthis invention are selected from the group consisting of hormones,cytokines and growth factors. Most preferred MPSFs for inducing boneand/or cartilage formation in concert with an osteogenic proteincomprise at least one compound selected from the group consisting ofinsulin-like growth factor I (IGF-I), estradiol, fibroblast growthfactor (FGF), growth hormone (GH), growth and differentiation factor(GDF), hydrocortisone (HC), insulin, progesterone, parathyroid hormone(PTH), vitamin D (1,25-(OH)₂D₃), retinoic acid and an interleukin,particularly IL-6.

[0141] In another preferred embodiment of this invention, the MPSFcomprises a compound or an agent that is capable of increasing thebioactivity of another MPSF. Agents that increase MPSF bioactivityinclude, for example, those that increase the synthesis, half-life,reactivity with other biomolecules such as binding proteins andreceptors, or the bioavailability of the MPSF. These agents may comprisehormones, growth factors, peptides, cytokines, carrier molecules such asproteins or lipids, or other factors that increase the expression or thestability of the MPSF.

[0142] For example, when the selected MPSF is IGF-I, agents thatincrease its bioactivity include GH, PTH, vitamin D, and cAMP inducers,which may thus function as MPSFs according to this invention. Inaddition, almost all of the IGF-I in circulation and the extracellularspace is bound by a group of high affinity binding proteins calledIGFBPs which can augment or inhibit IGF-I bioactivity (see, e.g., Jonesand Clemmons, Endocrine Reviews, 16, pp. 3-34 (1995)). Thus IGFBPs andagents which alter the levels of IGFBPs such that the bioactive IGF-Iconcentration is ultimately increased will also function as a MPSFaccording to this invention.

[0143] These or other agents that increase IGF-I bioactivity may be usedalone as the primary MPSF, or one or more may be used as additionalMPSFs in combination with IGF-I, to stimulate the tissue inductiveactivity of the morphogenic protein. One such preferred combinationcomprising at least two MPSFs for cartilage and bone formation isosteogenic protein OP-1, IGF-I and PTH.

[0144] Preferably, the MPSF is present in an amount capable ofsynergistically stimulating the tissue inductive activity of themorphogenic protein in a mammal. The relative concentrations ofmorphogenic protein and MPSF that will optimally induce tissue formationwhen administered to a mammal may be determined empirically by theskilled practitioner using the procedures described herein.

[0145] Implant Device

[0146] The invention also relates to an implant device for promotingbone formation, regeneration and repair. The implant device comprisesthe porous β-TCP material of the invention, and optionally at least onebioactive agent.

[0147] The implant device comprising the porous β-TCP material serves asa temporary scaffold and substratum for recruitment of migratoryprogenitor cells, and as a base for their subsequent anchoring andproliferation.

[0148] In a preferred embodiment, the implant device comprises theporous β-TCP matrix and a bioactive agent, which is dispersed orabsorbed in the matrix. It is envisioned that the bioactive agent caninclude but is not limited to bone morphogenic proteins, growth factorssuch as EGF, PDGF, IGF, FGF, TGF-α and TGF-β, cytokines, MPSF, hormones,peptides, lipids, trophic agents and therapeutic compositions includingantibiotics and chemotherapeutic agents, insulin, chemoattractant,chemotactic factors, enzymes, enzyme inhibitors. It is also envisionedthat bioactive agents such as vitamins, cytoskeletal agents, autograft,allograft, cartilage fragments, living cells such as chondrocytes, bonemarrow cells, mesenchymal stem cells, tissue transplants,immuno-suppressants may be added to the porous β-TCP.

[0149] The porous β-TCP matrix provides a sustained delivery or supportsystem for the bioactive agent, which is released over time at theimplantation site as the matrix material is slowly absorbed. In apreferred embodiment, the bioactive agent is encapsulated in thebiodegradable agent. The resorption of the biodegradable agent and thegradual release of the bioactive agent provides a sustained releasesystem. The dosage and rate of delivery of the bioactive agent may becontrolled based on the nature of the porous matrix, the nature of thebiodegradable agent and the nature of the binding interaction betweenthe bioactive agent encapsulated in the biodegradable agent, the porousmatrix and biodegradable agent. In a preferred embodiment, the bioactiveagent is a bone morphogenic protein or a nucleic acid molecule thatencodes BMP. In a most preferred embodiment, the BMP is OP-1.

[0150] In a preferred embodiment, the bioactive agent is a BMP. In amore preferred embodiment, the BMP is OP-1. The porous β-TCP matrix canprotect the BMP and MPSF from non-specific proteolysis, and canaccommodate each step of the cellular responses involved in progenitorcell induction during tissue development.

[0151] Studies have shown that the methodology for combining matrix andmorphogenic proteins plays a role in achieving successful tissueinduction. The optimal ratios of morphogenic protein to MPSF for aspecific combination and tissue type may be determined empirically bythose of skill in the art. Greater amounts may be used for largeimplants. The procedures used to formulate BMP and MPSF into the matrixare sensitive to the physical and chemical state of both the proteinsand the matrix.

[0152] In the preferred osteogenic device with porous β-TCP, theosteogenic protein diffuses out of the matrix into the implantation siteand permits influx and efflux of cells. The osteogenic protein inducesthe progenitor cells to differentiate and proliferate. Progenitor cellsmay migrate into the matrix and differentiated cells can move out of theporous matrix into the implant site. The sequential cellular reactionsin the interface of the bone matrix/osteogenic protein implants include:binding of fibrin and fibronectin to implanted matrix, migration andproliferation of mesenchymal cells, differentiation of the progenitorcells into chondroblasts, cartilage formation, cartilage calcification,vascular invasion, bone formation, remodeling, and bone marrowdifferentiation. The preferred osteogenic device with porous β-TCPmaterial, can be applied to bone formation in various orthopedic,periodontal, and reconstructive procedures.

[0153] The implant device may also comprise a binder in an admixturewith the bioactive agent and/or porous β-TCP material. The binder isadded to form a moldable putty which may be shaped to fit a defect siteor to take the form of a new tissue. The moldable putty composition canbe held in place by the surrounding tissue or masticated muscle. It ispreferred to shape the matrix to span a tissue defect and to take thedesired form of the new tissue. In the case of bone repair of anon-union defect, for example, it is desirable to use dimensions thatspan the non-union. Rat studies show that the new bone is formedessentially having the dimensions of the device implanted. Thus, thematerial may be used for subcutaneous or intramuscular implants. In boneformation procedures, the material is slowly absorbed by the body and isreplaced by bone in the shape of or very nearly the shape of theimplant.

[0154] Prosthetic Device

[0155] It is also contemplated that the porous β-TCP material of thepresent invention may be used in a prosthetic device. The prostheticdevice comprises a surface region that can be implanted adjacent to atarget tissue of a mammal, and a composition that is disposed on thesurface region. The prosthetic devices will be useful for repairingorthopedic defects, injuries or anomalies in the treated mammal.Preferably, the mammal is a human patient. The prosthetic device may bemade from a material comprising metal, ceramic or polymer compositematerial. Preferred devices comprise a load-bearing core selected fromCo—Cr—Mo alloys, titanium alloys and stainless steel. Preferredprosthetic devices are selected from the group consisting of a hipdevice, a fusion cage and a maxillofacial device.

[0156] The composition comprises the porous β-TCP material of theinvention, and optionally, one or more agents selected from the groupconsisting of a bioactive agent or a binder dispersed in the porousβ-TCP. In a preferred embodiment, the bioactive agent is encapsulated inthe biodegradable agent. In a preferred embodiment, the bioactive agentis a BMP or nucleic acid encoding BMP, more preferably, OP-1. Osteogenicprotein-coated prosthetic devices may enhance the bond strength betweenthe prosthesis and existing bone. (Rueger et al., U.S. Pat. No.5,344,654, incorporated herein by reference). The composition may act asa coating for synthetically constructed bone material, such as for anartificial hip, replacement of diseased bone, correction of defects, oranchoring teeth. The composition is disposed on the surface of theimplant in an amount sufficient to promote enhanced tissue growth intothe surface. The amount of the composition sufficient to promoteenhanced tissue growth may be determined empirically by those of skilledin the art using bioassays described in Rueger et al., U. S . Pat. No.5,344,654, incorporated herein by reference. Preferably, animal studiesare performed to optimize the concentration of the compositioncomponents before a similar prosthetic device is used in the humanpatient.

[0157] In another preferred embodiment, the composition is applied tothe clinical procedure of total joint arthroplasty in hips, knees,elbows and other joints, wherein a diseased or damaged natural joint isreplaced by a prosthetic joint. For example, in a total hiparthroplasty, an acetabular cup is inserted with the composition in theacetabular socket of the pelvis to replace the natural acetabulum. Thecup is held in place by the composition and secured by fixation screws.Generally, the cavity or socket conforms to the outer surface of theacetabular cup. The composition can also be applied to total jointrevision surgery, to strengthen the bondage between joint prostheticdevices and the bone.

[0158] In yet another preferred embodiment, the composition is appliedto a clinical procedure called vertebroplasty. The composition isinjected into the interior of a vertebral body. This method is used inthe treatment of osteoporosis to increase the density of bone.

[0159] In a preferred embodiment, the prosthetic device is selected fromthe group consisting of a fusion cage, a dowel and other devices havinga pocket or chamber, such as an interbody fusion for containing thecomposition of the present invention. Preferably, the interbody fusiondevice is produced from material selected from the group consisting oftitanium, PEEK (poly(etheretherketone)) and allograft. The interbodyfusion in the cervical, thoracic and lumbar spine can be administeredvia an anterior or posterior approach. Alternatively, the composition ofthis invention can be used without an associated interbody device toachieve interbody fusion.

[0160] Spinal fusion cages are placed into the intervertebral space leftafter the removal of a damaged spinal disc to eliminate local motion andto participate in vertebral to vertebra bony fusion. As described inU.S. Pat. No. 5,015,247, incorporated herein by reference, the fusioncages are in the form of a cylindrical hollow member having an outsidediameter larger than the space between two adjacent vertebrae to befused. The interior space within the cylindrical hollow implant can befilled with the composition of this invention. The cylindrical implantscan also include a threaded exterior to permit threaded insertion into atapped bore formed in the adjacent vertebrae. Alternatively, some fusionimplants have been designed to be impacted into the intradiscal space.As described in U.S. Pat. No. 6,146,420, incorporated herein byreference, the fusion device includes opposite end pieces with anintegral central element. The central element has a much smallerdiameter so that the fusion device forms an annular pocket around thecentral element. The composition of this invention can be disposedwithin the annular pocket between the opposite end pieces.

[0161] In a preferred embodiment, the prosthetic device is used forrepair of osseous and discoligamentous instability. The composition ofthis invention may be applied to the intervertebral area, resulting insuperior fusion and consequently achieving definitive stabilization of atraumatized motor segment via a single dorsal approach. This applicationmay eliminate the need to undergo a second operation for fractures ofthe thoracolumbar spine, which, at present, is often necessary butinvolves additional high risks. Also, this method avoids the problemsassociated with transplantation of autogenous cancellous bone and itsassociated risk of high morbidity might be avoided. See, e.g., Rueger etal., Orthopäde, 27, pp. 72-79 (1998).

[0162] In another preferred embodiment, the prosthetic device is amaxillofacial device. Maxillofacial devices are applied externally tocorrect facial defects resulting from cancer surgery, accidents,congenital deformities. In order to restore the masticatorydeficiencies, a patient with marginal bone mass is first treated withthe composition of this invention to pack and build up the surgicalsite. A maxillofacial anchoring and distracting system, as illustratedin U.S. Pat. No. 5,899,940, incorporated herein by reference, can beapplied to increase the existing bone quality. Fixation devices, such asa standard threaded bone screw and simple pin point tack or self-lockingand threaded bone tack screw device (U.S. Pat. No. 5,971,985,incorporated herein by reference), are used for the retention of tissuegrafts and synthetic membranes to the maxillofacial bone graft site.Once the site has healed, a second surgery is performed to insert theappropriate length endosseous dental implant and to restore masticatoryfunction.

[0163] The invention also provides a method for promoting in vivointegration of an implantable prosthetic device of this invention into atarget tissue of a mammal comprising the steps of a) providing on asurface of the prosthetic device a composition comprising the porousβ-TCP material, optionally, at least one bioactive agent or a binder,and b) implanting the device in a mammal at a locus where the targettissue and the surface of the prosthetic device are maintained at leastpartially in contact for a time sufficient to permit tissue growthbetween the target tissue and the device.

[0164] Method of Inducing Bone Formation and Delivery

[0165] The invention also provides a method of inducing bone formationin a mammal. The mammal is preferably a human patient. The methodcomprises the step of implanting in the defect site of a mammal acomposition comprising the porous β-TCP of the invention. In a preferredembodiment, the composition may further comprise a binder and/or abioactive agent. The defect can be an endochondreal defect, anosteochondral defect or a segmental defect. The method can be applied toother defects which are not limited to, non-union fractures; bonecavities; tumor resection; fresh fractures (distracted or undistracted);cranial, maxillofacial and facial abnormalities, for example, in facialskeletal reconstruction, specifically, orbital floor reconstruction,augmentation of the alveolar ridge or sinus, periodontal defects andtooth extraction socket; cranioplasty, genioplasty, chin augmentation,palate reconstruction, and other large bony reconstructions;vertebroplasty, interbody fusions in the cervical, thoracic and lumbarspine and posteriolateral fusions in the thoracic and lumbar spine; inosteomyelitis for bone regeneration; appendicular fusion, ankle fusion,total hip, knee and joint fusions or arthroplasty; correcting tendonand/or ligamentous tissue defects such as, for example, the anterior,posterior, lateral and medial ligaments of the knee, the patella andachilles tendons, and the like as well as those defects resulting fromdiseases such as cancer, arthritis, including osteoarthritis, and otherbone degenerative disorders such as osteochondritis dessicans. Themethod may be used in bone augmentation, bone prosthesis, hard tissueimplant, bone scaffolding, fixation systems (e.g. screws, sutures,suture anchors, staples, surgical tacks, clips, plates and screws).

[0166] The invention also provides a method of delivering a bioactiveagent at a site requiring bone formation comprising the step ofimplanting the porous β-TCP and a bioactive agent at the defect site ofa mammal. The method of delivering the bioactive agent may furtherinclude a binder. In a preferred embodiment, the bioactive agent isencapsulated in a biodegradable agent. In a preferred embodiment, thebioactive agent belongs to the bone morphogenic protein family. Inanother preferred embodiment, the bioactive agent is a nucleic acidmolecule comprising a sequence encoding a BMP. Preferably, the nucleicacid is trapped in a carrier. In yet another embodiment, the bioactiveagent is a bone cell or a cell transfected with nucleic acid encodingBMP. In another preferred embodiment, the delivery of the bioactiveagent is sustained release. The biodegradable agent is preferably abiocompatible and non-immunogenic polymer, more preferably, PLGA. Thebioactive agent is preferably OP-1. The release rate of the bioactiveagent can be controlled by altering the molecular weight of the PLGA.The degradation of PLGA commences when water penetrates the cementmatrix to hydrolyze long polymer chains into short water solublefragments. This results in a reduction in the molecular weight of thePLGA without loss in its physical properties. Gradually, further erosionof the polymer leads to the disruption of the polymer, thereby releasingthe bioactive agent. For example, in the case of 10 kD to 30 kD PLGA,the rate of release for OP-1 is one to six weeks.

[0167] The invention also describes a method of delivering a bioactiveagent at a site requiring cartilage formation comprising implanting atthe defect site of a mammal a composition comprising the bioactive agentand biodegradable agent, wherein the bioactive agent is encapsulated inthe biodegradable agent. Preferably, the bioactive agent is OP-1 and thebiodegradable agent is PLGA.

EXAMPLE 1 Preparation of Tricalcium Phosphate

[0168] A slurry of lime (calcium oxide-hydroxide) is prepared and dilutephosphoric acid is added dropwise to the slurry, which is stirredconstantly. The molar proportion of calcium oxide to phosphoric acid is3:2. The product characteristics are evaluated by X-ray diffraction andadjustments are made to the proportions if required. The resultantslurry is harvested by spray drying. If the slurry is harvested byfiltration, the dried cake is crushed to a fine powder of amorphous TCP.The particle size of the amorphous TCP is preferably smaller than 10 μm.

EXAMPLE 2 Preparation of β-TCP Granule

[0169] The TCP powder was mixed with polystyrene beads (NUNCA/S-Denmark)(0-160 μm beads). The 10% polyvinyl pyrrolidone (PVP)granulating solution was prepared by adding PVP C-30 (Plasdone C-30, ISPtechnologies lot # TX 60810) in small portions in a beaker or flask ofstirring water until the solution was clear. About 37 ml of 10% PVPsolution was added to the TCP mixture in 5 ml increments to form acrumbly mass. As illustrated in Table 1, mixtures were prepared withdifferent proportions of pore-forming beads and TCP. TABLE 1 beadcomposition (w/w) beads (g) TCP (g) 12.5% 12.5 87.5   25% 12.5 37.537.5% 18.75 31.25   50% 23.75 23.75

[0170] The crumbly mass was passed through <500 μm, 500-1000 μm, or1000-1700 μm sieves under a vibrating motion to produce wet granuleshaving the corresponding particle size ranges. The sieved material wasdried under vacuum at 105° C. for 2-3 hours.

[0171] The dried granules then underwent a burn off cycle tovaporize/carbonize the pore-forming material and were subsequentlysintered at 1150° C. The temperature was raised from 39° C. to 300° C.over an 18 hour period, held at 300° C. for 1 hour, elevated to 700° C.over an 18 hour period, held at 700° C. for 2 hours, and elevated to1150° C. over a 6 hour period, and held at 1150° C. for 6 hours, andslow cooled to 39° C. over a 6 hour period. After the sintering cycle,the resultant material was confirmed by X-ray diffraction to be porouscrystalline @-TCP.

[0172] The 37.5% w/w, 500-1000 μm sintered granules were resieved andmixed with the binder, carboxy methylcellulose sodium to form a moldableputty. The putty mixtures were formed with different proportions ofβ-TCP and CMC. All combinations of β-TCP and CMC produced a putty havingappropriate adherence properties, and did not break up in excess water.The cohesiveness of the putty was enhanced as the CMC proportionincreased. The β-TCP/CMC 1:0.4 (w/w) putty showed the bestcharacteristics for handling. The rheological properties of the varioussamples were determined.

EXAMPLE 3 Rat Model Bioassay for Bone Induction

[0173] This assay consists of implanting samples in subcutaneous sitesin recipient rats under ether anesthesia. Male Long-Evans rats, aged28-32 days, may be used. A vertical incision (1 cm) is made understerile conditions in the skin over the thoracic region, and a pocket isprepared by blunt dissection. Approximately 25 mg of the test sample isimplanted deep into the pocket and the incision is closed with ametallic skin clip. The day of implantation is designated as day one ofthe experiment. Implants are removed at varying times thereafter (i.e.12 days, 18 days). The heterotrophic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotropic sites.

[0174] Bone growth is determined biochemically by calcium content of theimplant. Calcium content, is proportional to the amount of bone formedin the implant. Bone formation therefore is calculated by determiningthe calcium content of the implant in rats and is expressed as “boneforming units,” where one bone forming unit represents the amount ofprotein that is needed for half maximal bone forming activity of theimplant. Bone induction exhibited by intact demineralized rat bonematrix is considered to be the maximal bone differentiation activity forcomparison purposes in this assay.

[0175] Cellular Events During Endochondral Bone Formation

[0176] Successful implants exhibit a controlled progression through thestages of protein-induced endochondral bone development, including: (1)transient infiltration by polymorphonuclear leukocytes; (2) mesenchymalcell migration and proliferation; (3) chondrocyte appearance; (4)cartilage matrix formation; (5) cartilage calcification; (6) vascularinvasion, appearance of osteoblasts, and formation of new bone; (7)appearance of osteoclasts, bone remodeling and dissolution of theimplanted matrix; and (8) hematopoietic bone marrow differentiation inthe ossicles. This time course in rats may be accelerated by increasingthe amounts of OP-1 added. It is possible that increasing amounts of oneor more MPSFs may also accelerate this time course. The shape of the newbone conforms to the shape of the implanted matrix.

[0177] Histological Evaluation

[0178] Histological sectioning and staining is preferred to determinethe extent of osteogenesis in the implants. Implants are fixed in BouinsSolution, embedded in paraffin, and cut into 6-8 μm sections. Stainingwith toluidine blue or hemotoxylin/eosin demonstrates clearly theultimate development of endochondral bone. Twelve-day implants areusually sufficient to determine whether the implants containnewly-induced bone.

[0179] Biological Markers

[0180] Alkaline phosphatase (AP) activity may be used as a marker forosteogenesis. The enzyme activity may be determinedspectrophotometrically after homogenization of the implant. The activitypeaks at 9-10 days in vivo and thereafter slowly declines. Implantsshowing no bone development by histology have little or no alkalinephosphatase activity under these assay conditions. The assay is usefulfor quantification and obtaining an estimate of bone formation quicklyafter the implants are removed from the rat. Alternatively, the amountof bone formation can be determined by measuring the calcium content ofthe implant.

[0181] Gene expression patterns that correlate with endochondral bone orother types of tissue formation can also be monitored by quantitatingmRNA levels using procedures known to those of skill in the art such asNorthern Blot analysis. Such developmental gene expression markers maybe used to determine progression through tissue differentiation pathwaysafter osteogenic protein/MPSF treatments. These markers includeosteoblastic-related matrix proteins such as procollagen α₂ (I),procollagen α₁ (I), procollagen α₁ (III), osteonectin, osteopontin,biglycan, and alkaline phosphatase for bone regeneration (see e.g., Suvaet al., J. Bone Miner. Res., 8, pp. 379-88 (1993); Benayahu et al., J.Cell. Biochem., 56, pp. 62-73 (1994)).

EXAMPLE 4 Sheep Model Bioassay for Bone Repair

[0182] Skeletally mature female sheep were included in the study. Threedrilled defects were created in the area of the proximal metaphysis forboth the left and right tibia of each animal. Defects were 6 mm indiameter and at least 10 mm deep. The defect size was consistent acrossall test animals. The defects were created so as to maintain thestructure of the interosseous fibrofatty marrow. This marrow acts as abarrier between the implant materials and prevents interosseous mixingof the matrix materials tested. As illustrated in Table 2, β-TCP puttyI, II, III, IV and collagen were tested in the defect sites with andwithout OP-1. OP-1 was either directly added to the β-TCP formulationsor encapsulated in PLGA. Table 3 represents examples of formulationswherein the OP-1 is encapsulated in PLGA. Of the six defect sites ineach animal, one defect site served as a control, which contained notest material.

[0183] A 3 to 4 inch incision was made over the proximal tibialmetaphysis. The skin and underlying muscle were dissected to expose theperiosteum. The periosteum was incised and maintained intact forsurgical closure if possible. Three transverse holes were created in themetaphysis. The first and most superior was created approximately 2 cmbelow the articular surface of the tibia. The defects were created so asto form a line oriented with the long axis of the bone. Implants werespaced at 1.6 cm intervals measured center-to-center.

[0184] Materials were harvested at four and eight weeks post-treatment.Animals were euthanised with pentobarbital 75-100 mg/kg IV. The proximaltibia were taken and cut to best allow for tissue fixation. Specimenswere fixed in 10% neutral buffered Formalin. Specimens were cut, iffeasible, so as to capture all implant sites in a single specimen.Following fixation, specimens were decalcified, embedded in plastic andsectioned in longitudinal orientation using Exackt technique and groundto appropriate section thickness for histologic interpretation.

[0185] Radiographic assessment (FIGS. 9-16, 27 and 28) and histologicevaluation (FIGS. 1-8) were made at post-operative, four and eight weekson all implant sites. Anterior posterior radiographs were taken so as tobest image all three defects simultaneously and view the cylindricaldefects from the side. Qualitative histologic descriptions identifiednew bone formation, residual implant material and any evidence ofpathologic response. Images were captured for each specimen and scorespresented for bone formation, acute and chronic inflammation andresidual matrix.

[0186] Specimen handling and hemostatic properties were recorded at thetime of implantation. Materials ranged in form and consistency from aputty or granular form to a semi-solid cylinder. TABLE 2 Initialpore-former Code Formulation percentage/Granule size 89A β-TCP Putty I12.5% (w/w), 0.5-1 mm 89B β-TCP Putty II 25% (w/w), 0.5-1 mm 89C β-TCPPutty III 37.5% (w/w), 0.5-1 mm 89F β-TCP Putty IV 25% (w/w), 1-2 mm 48CCollagen Bovine type I collagen SOB1 Lyophil 1 OP-1 SOP2 Lyophil 2Placebo Reconstitution Resconstitution medium

[0187] TABLE 3 Code Formulation formulation 4 β-TCP, 7% (w/w) PLGA (10kD) with 0.3% (w/w) OP-1 formulation 5 β-TCP, 7% (w/w) PLGA (25-30 kD)with 0.3% (w/w) OP-1

[0188] Formulation Handling

[0189] Lyophil 1 and Lyophil 2 (placebo) were reconstituted by adding2.5 ml of the reconstitution medium to one vial of the Lyophil (Allcomponents were stored frozen at 2 to 8° C. until use), shaking themedium gently for 2 minutes until a homogenous (clear to cloudy) gel wasformed. 0.4 ml of reconstituted Lyophil gel was added to the porousβ-TCP matrix slowly and with care. Utilizing a thin spatula, the porousβ-TCP matrix was mixed with the gel to form a putty-like material.

[0190] The PLGA microspheres (particle size 75-150 μm, Alkermes, Inc.)encapsulated with 0.3% (w/w) OP-1 were mixed with the porous β-TCPmatrix.

[0191] The putty material was immediately implanted. The implantmaterials were placed through the use of a folded piece of sterilepaper. The paper was filled with test material and used to pour it intothe defect while continuously packing material in the site. The handlingproperties prior to placement and in the defect site were recorded.

[0192] The β-TCP Putty I, II, III, IV formulations were poured as agranular dry powder. Once combined with the vehicle solution, theputties had a dry crunchy granular texture. The formulations absorbedall of the Lyophil solution. The formulation was implanted with aspatula. Once in the implant site, the materials became well filled withblood.

[0193] The collagen formulation poured as a fluffy powder. Once mixedwith a vehicle solution, it had a gritty putty texture. The formulationcould be easily placed with a syringe in the implant site. The implantsite became well filled with blood.

[0194] Histologic Results

[0195] Proximal tibia sections contained three defects. These defectswere gross macro-cut so that all three were contained in a singlesection. Based on gross section observations, clinical assays, andfaxitron x-rays of this section, the section was consideredrepresentative of the sample. This orientation allowed the evaluation ofthe periosteal reaction overlying the defects and intramedullaryresponse to the test materials. Specimens were evaluated from 4 and 8week explants (FIGS. 1-8). All three defects within a single tibialsection received either the placebo or OP-1 solution. This segregationof the placebo and OP-1 implants facilitated the determination of theactive or inactive biologic nature of the implant material.

[0196] Four-Week Evaluation for OP-1 and Placebo Implant Materials

[0197] At four weeks, the β-TCP Putty I (89A) was present in all sites(FIG. 3 middle site and FIG. 2 distal site). Generally, the matrix wasnot significantly resorbed nor was it undergoing active resorption.Sites treated with OP-1 resulted in some but not marked new boneformation (FIG. 3 middle site). Placebo treated sites had bone formationat the level of the cortex (FIG. 2 distal site).

[0198] The β-TCP Putty II (89B) was present in all sites at 4 weeks insignificant amounts (FIG. 3 distal site and FIG. 1 proximal site). Therewas no significant evidence of matrix resorption. OP-1 treated sitesresulted in small amounts of new bone formation predominately at thecortical and periosteal level (FIG. 3 distal site). Of the four β-TCPputty formulations tested, β-TCP putty II resulted in more inflammationthan the other three formulations. Foreign body giant cells (FBGC) werereported in conjunction with this inflammation.

[0199] β-TCP Putty III (89C) was present in significant amounts in allsix sites treated at 4 weeks (FIG. 1 middle site and FIG. 4 proximalsite). OP-1 treatment did not noticeably alter residual matrix volumes.Bone formation at the cortical level was apparent in OP-1 treatedspecimens (FIG. 4 proximal site) and less common in placebo treatedsites (FIG. 1 middle site). Little or no inflammation was observed inresponse to the β-TCP matrix independent of OP-1 treatment.

[0200] β-TCP Putty IV (89F) was present in significant amounts in allsix sites treated at 4 weeks (FIG. 1 distal site and FIG. 4 middlesite). OP-1 treatment had no apparent effect on residual matrix volume.OP-1 treated sites resulted in greater bone formation throughout thematrix with cortical and periosteal responses apparent (FIG. 4 middlesite). Little or no inflammation was observed in response to the β-TCPmatrix independent of OP-1 treatment.

[0201] Eight-Week Evaluation for OP-1 and Placebo Treated ImplantMaterials

[0202] The β-TCP Putty I (89A) was present in all sites at 8 weeks (FIG.7 proximal site and FIG. 6 distal site). The OP-1 treated implantsgenerally showed evidence of a strong bone inductive response (FIG. 7proximal site). In two OP-1 treated sites, the β-TCP matrix appeared tohave significantly degraded. Sites treated with OP-1 resulted in markednew bone formation at the cortical level with modest bone infiltrationinto the matrix within the medullary space. Placebo treated sitesresulted in less bone formation at the level of the cortex (FIG. 6distal site).

[0203] The β-TCP Putty II (89B) was present in all sites at 8 weeks insignificant amounts (FIG. 5 proximal site and FIG. 7 middle site). Therewas no significant evidence of matrix resorption. OP-1 treated sitesresulted in small amounts of new bone formation predominately at thecortical and periosteal level and closure at the defect site (FIG. 7middle site). Placebo treated materials resulted in less bone formationat the cortical level and calcium particles blocking closure of thecortical defect (FIG. 5 proximal site). The inflammation notedpreviously in response to this material was not evident.

[0204] β-TCP Putty III (89C) was present in significant amounts in allsix sites treated at 8 weeks (FIG. 5 middle site and FIG. 7 distalsite). OP-1 treatment did not noticeably alter residual matrix volumes.Bone formation at the cortical level and a marked periosteal responsewas apparent in OP-1 treated specimens (FIG. 7 distal site). Little orno inflammation was observed in response to the β-TCP matrix independentof OP-1 treatment.

[0205] β-TCP Putty IV (89F) was present in significant amounts in allsix sites treated at 8 weeks (FIG. 5 distal site and FIG. 8 proximalsite). A few sites had less residual matrix than others. Generally, OP-1treatment had no apparent effect on residual matrix volume. OP-1 treatedsites resulted in greater bone formation throughout the matrix with anapparent cortical and periosteal response (FIG. 8 proximal site). Littleor no inflammation was observed in response to the β-TCP matrixindependent of OP-1 treatment.

[0206] Conclusion of the Above Results

[0207] Compared to the collagen material which demonstrated acute andchronic inflammation coupled with an FBGC response, the four porousβ-TCP formulations resulted in little or no inflammation at four andeight weeks. OP-i treatment in the porous β-TCP materials consistentlyexhibited marked bone formation at the cortical level and a reactiveperiosteal response that often resulted in cortical defect closure.Although the large granular (1-2 mm) β-TCP putty IV formulation appearedto allow bone ingrowth deeper in the matrix, there was greaterinter-granular spacing compared to that observed in small granular β-TCPputties.

[0208] Paraffin Histology Study

[0209] Tissues from the sheep model bioassay were evaluated usingparaffin sections and hematoxylin and eosin stain to evaluate the effectof particle size and porosity of the implant material on bone formationin and around particles.

[0210] Tibial specimens were sectioned so as to isolate implant sites inthe proximal, middle and distal sites from four animals (138, 299, 297,and 295). These explants were decalcified, embedded in paraffin,sectioned and stained with hematoxylin and eosin.

[0211] Sections were viewed using light microscopy and interpreted forthe effect of particle size and porosity. For specimens stratified inbone formation, the response from the cortical level was robust anddeep, and the response was modest in the medullary compartment. Due tothis stratification, the level extending from the endosteal cortex to alevel 2-3 mm deep was evaluated.

[0212] Each of the four ceramic formulations were evaluated for boneformation in the pores and bone bridging across the particles. Boneformation in pores was assessed by counting pores that were completelyisolated within a particle from the adjacent stroma. Pores that wereobvious and generally round were counted. As pores were counted, a ratiowas formed of those that had bone over those that did not. This is notedas the pore-fill ratio.

[0213] Pore counting was performed by scanning the field. In materialswith few pores, the majority were counted as the field was scanned (FIG.25). In materials with many pores, regions were counted and a new regionwas viewed and then counted (FIG. 26). The average of the regions ortotal count were presented in the ratio.

[0214] Bone bridging between particles was scored 0 to 2. A zero scorewas given to particles when the bone did not bridge to adjacentparticles. A score of 1 was given when a couple to a few particlesconsistently showed bridging. A score of 2 was given when many of theparticles were joined by vital bone trabeculae.

[0215] Tables 4 and 5 illustrate the pore-fill ratios and bone bridgingscores for placebo and OP-1 at four weeks (FIGS. 17-20). Tables 6 and 7illustrate the pore-fill ratios and bone bridging scores for placebo andOP-1 at eight weeks (FIGS. 21-24). Bone bridging was more pronounced forβ-TCP putties made from 37.5% (w/w) pore-forming agent and having thesmaller 0.5-1 mm granule size(Tables 4-7). The pore-fill ratio wasgenerally equivalent for the β-TCP putty made from 25% and 37.5% (w/w)pore-forming agents. The β-TCP made from 12.5% (w/w) pore-forming agenthad a lower pore-fill ratio (Tables 4-7). The pore-fill ratio wasconsistently higher in the 89F formulation due to the larger size of theparticle (1-2 mm) with more pores per particle. Compared to the smallparticles (0.5-1 mm), there was less bone bridging in the largerparticles due to the fact that more bone was required to bridge largeparticles. TABLE 4 Initial Pore Bone Particle Pore- Duration Fill Bridg-Section Treatment Size former % (wks) Ratio ing 297R-D 89A .5-1 mm 12.54  2/10 0 297L-P 89B .5-1 mm 25 4  6/10 0 297L-M 89C .5-1 mm 37.5 4 6/70 297L-D 89F  1-2 mm 25 4 10/10 0

[0216] TABLE 5 Initial Pore Bone Particle Pore- Duration Fill Bridg-Section Treatment Size former % (wks) Ratio ing 295L-M 89A .5-1 mm 12.54  6/11 2 295L-D 89B .5-1 mm 25 4  8/11 1 295R-P 89C .5-1 mm 37.5 4 6/82 295R-M 89F  1-2 mm 25 4 10/10 2

[0217] TABLE 6 Initial Pore Bone Particle Pore- Duration Fill Bridg-Section Treatment Size former % (wks) Ratio ing 299R-D 89A .5-1 mm 12.58  4/14 1 299L-P 89B .5-1 mm 25 8  9/10 2 299L-M 89C .5-1 mm 37.5 818/20 2 299L-D 89F  1-2 mm 25 8  9/10 1

[0218] TABLE 7 Initial Pore Bone Particle Pore- Duration Fill Bridg-Section Treatment Size former % (wks) Ratio ing 138L-P 89A .5-1 mm 12.58 10/20 1 138L-M 89B .5-1 mm 25 8 8/9 2 138L-D 89C .5-1 mm 37.5 8 10/122 138R-P 89F  1-2 mm 25 8  9/10 1

[0219] Conclusion of Paraffin Histology Study

[0220] For β-TCP formulations, bone formation in pores became moreapparent as the porosity increased. Bone formation in pores was lessfrequent in the material made from 12.5% pore-former compared to thematerial made from 37.5% pore-former. Although bone formation was moreobvious in larger particles (1-2 mm), less bone bridging was observed inthese large particles.

[0221] The collagen formulations resulted in no bone formation and amarked pathologic response. Moreover, these formulations resulted in amarked FBGCR and chronic fibroinflammatory response.

EXAMPLE 5 Feline Model Bioassay for Bone Repair

[0222] A femoral osteotomy defect is surgically prepared. Withoutfurther intervention, the simulated fracture defect would consistentlyprogress to non-union. The effects of osteogenic compositions anddevices implanted into the created bone defects are evaluated by thefollowing study protocol.

[0223] Briefly, the procedure is as follows: Sixteen adult cats eachweighing less than 10 lbs. undergo unilateral preparation of a 1 cm bonedefect in the right femur through a lateral surgical approach. In otherexperiments, a 2 cm bone defect may be created. The femur is immediatelyinternally fixed by lateral placement of an 8-hole plate to preserve theexact dimensions of the defect. Four different types of materials may beimplanted in the surgically created cat femoral defects: group I is anegative control group with no test material; group II is implanted withbiologically active porous β-TCP; group III is implanted with porousβ-TCP and an osteogenic protein; and group IV is implanted with porousβ-TCP, an osteogenic protein and MPSF.

[0224] All animals are allowed to ambulate ad libitum within their cagespost-operatively. All cats are injected with tetracycline (25 mg/kgsubcutaneously (SQ) each week for four weeks) for bone labeling.

[0225] In vivo radiomorphometric studies are carried out immediately at4, 8, 12 and 16 weeks post-operative by taking a standardized X-ray ofthe lightly-anesthetized animal positioned in a cushioned X-ray jigdesigned to consistently produce a true anterio-posterior view of thefemur and the osteotomy site. All X-rays are taken in exactly the samefashion and in exactly the same position on each animal. Bone repair iscalculated as a function of mineralization by means of random pointanalysis. A final specimen radiographic study of the excised bone istaken in two planes after sacrifice.

[0226] Excised test and normal femurs may be immediately studied by bonedensitometry, or wrapped in two layers of saline-soaked towels, placedinto sealed plastic bags, and stored at −20° C. until further study.Bone repair strength, load-to-failure, and work-to-failure are tested byloading to failure on a specially designed steel 4-point bending jigattached to an Instron testing machine to quantitate bone strength,stiffness, energy absorbed and deformation to failure. The study of testfemurs and normal femurs yields the bone strength (load) in pounds andwork-to-failure in joules. Normal femurs exhibit a strength of 96(+/−12) pounds. Osteogenic device-implanted femur strength should becorrected for surface area at the site of fracture (due to the“hourglass” shape of the bone defect repair). With this correction, theresult should correlate closely with normal bone strength.

[0227] Following biomechanical testing, the bones are immediately slicedinto two longitudinal sections at the defect site, weighed, and thevolume measured. One-half is fixed for standard calcified bonehistomorphometrics with fluorescent stain incorporation evaluation, andone-half is fixed for decalcified hemotoxylin/eosin stain histologypreparation.

[0228] Selected specimens from the bone repair site are homogenized incold 0.15 M NaCl, 3 mM NaHCO₃, pH 9.0 by a Spex freezer mill. Thealkaline phosphatase activity of the supernatant and total calciumcontent of the acid soluble fraction of sediment are then determined.

EXAMPLE 6 Rabbit Model Bioassay for Bone Repair

[0229] This assay is described in detail in Oppermann et al., U.S. Pat.No. 5,354,557; see also Cook et al., J. of Bone and Joint Surgery, 76-A,pp. 827-38 (1994), which are incorporated herein by reference). Ulnarnon-union defects of 1.5 cm are created in mature (less than 10 lbs) NewZealand White rabbits with epiphyseal closure documented by X-ray. Theexperiment may include implantation of devices into at least eightrabbits per group as follows: group I negative control implants withouttest material; group II implants with porous β-TCP; group III implantswith porous β-TCP and an osteogenic protein; group IV implants withporous β-TCP, osteogenic protein and MPSF combinations. Ulnae defectsare followed for the full course of the eight week study in each groupof rabbits.

[0230] In another experiment, the marrow cavity of the 1.5 cm ulnardefect is packed with activated osteogenic protein in porous β-TCP inthe presence or absence of a MPSF. The bones are allografted in anintercalary fashion. Negative control ulnae are not healed by eightweeks and reveal the classic “ivory” appearance. In distinct contrast,the osteogenic protein/MPSF-treated implants “disappear”radiographically by four weeks with the start of remineralization by sixto eight weeks. These allografts heal at each end with mildproliferative bone formation by eight weeks. This type of device servesto accelerate allograft repair.

[0231] Implants treated with osteogenic protein in the presence of aMPSF may show accelerated repair, or may function at the same rate usinglower concentrations of the osteogenic protein. As was described above,the rabbit model may also be used to test the efficacy of and tooptimize conditions under which a particular osteogenic protein/MPSFcombination can induce local bone formation.

EXAMPLE 7 Dog Ulnar Defect Bioassay For Bone Repair

[0232] This assay is performed essentially as described in Cook et al.,Clinical Orthopaedics and Related Research, 301, pp. 302-112 (1994),which is incorporated herein by reference). Briefly, an ulnar segmentaldefect model is used to evaluate bone healing in 35-45 kg adult maledogs. Experimental composites comprising 500 mg of porous β-TCP arereconstituted with varying amounts of OP-1 in the absence or presence ofincreasing concentrations of one or more putative MPSFs. Any osteogenicprotein may be used in place of OP-1 in this assay. Implantations atdefect sites are performed with one carrier control and with theexperimental series of OP-1 and OP-1/MPSF combinations being tested.Mechanical testing is performed on ulnae of animals receiving compositesat 12 weeks after implantation. Radiographs of the forelimbs areobtained weekly until the animals are sacrificed at either 12 or 16postoperative weeks. Histological sections are analyzed from the defectsite and from adjacent normal bone.

[0233] The presence of one or more MPSFs may increase the rate of bonerepair in dog. The presence of one or more MPSFs may also permit the useof reduced concentrations of osteogenic protein per composite to achievesimilar or the same results.

EXAMPLE 8 Monkey Ulnar and Tibial Defect Bioassay for Bone Repair

[0234] This bone healing assay in African green monkeys is performedessentially as described in Cook et al., J. Bone and Joint Surgery, 77A,pp. 734-50 (1995), which is incorporated herein by reference. Briefly, a2.0 cm osteoperiosteal defect is created in the middle of the ulnarshaft and filled with an implant comprising porous β-TCP matricescontaining OP-1 in the absence or presence of increasing concentrationsof one or more putative MPSFs. Experimental composites comprising porousβ-TCP matrices reconstituted with varying amounts of OP-1 in the absenceor presence of increasing concentrations of one or more putative MPSFsare used to fill 2.0 cm osteoperiosteal defects created in the diaphysisof the tibia. Any osteogenic protein may be used in place of OP-1 inthis assay. Implantations at defect sites are performed with one carriercontrol and with the experimental series of OP-1 and OP-1/MPSFcombinations being tested. Mechanical testing is performed on ulnae andtibia of animals receiving composites. Radiographs and histologicalsections are analyzed from the defect sites and from adjacent normalbone as described in Cook et al.

[0235] The presence of one or more MPSFs can increase the rate of bonerepair in the monkey. The presence of one or more MPSFs may also permitthe use of reduced concentrations of osteogenic protein per composite toachieve similar or the same results.

EXAMPLE 9 Goat Model Fracture Healing Bioassay

[0236] This fracture healing assay in sheep is performed essentially asdescribed in Blokhius et al., Biomaterials, 22, pp. 725-730 (2001),which is incorporated herein by reference. A closed midshaft fracture iscreated in the left tibia of adult female goats with a custom-made threepoint bending device. The fractures are stabilized with an externalfixator, which is placed at the lateral side of the tibia. Threedifferent types of materials are implanted in the goat defects viainjection: group I is a negative control group with no test material;group II is implanted with the biologically active porous β-TCP; groupIII is implanted with porous β-TCP and an osteogenic protein; and groupIV is implanted with porous β-TCP and an osteogenic protein encapsulatedin PLGA. The test material is placed in the fractured gap. Mechanicaltesting (four-point non-destructive bending test) is performed on theanimals receiving composites at two weeks and four weeks. After themechanical testing, anterior, posterior, lateral, and medial slices ofthe fracture gap are sawn to perform radiographs and histologicalsections.

EXAMPLE 10 Fusion Assay of an Unstable Motor Segment of the Sheep LumbarSpine

[0237] This assay investigates the healing of osseous anddiscoligamentous instability. A motor segment of the spine is afunctional unit consisting of two vertebral bodies lying one above theother, and an intervertebral disc.

[0238] A trial group consists of 12 sheep. Two control groups of 12sheep each are used. The surgical area at the inferior lumbar spine isprepared after introduction of general anesthesia and placing theanimals in prone position. A skin incision of about 12 cm in lengthabove the spinous processes of the inferior lumbar spine is made. Aftertranssection of the subcutis and fascia, the back muscles are moved tothe side.

[0239] Intubation anesthesia is applied by intramuscular injection of1.5 ml xylazine (Rompun®). Further dosage can be administered as needed.The sedation requires placement of an intravenous indwelling catheterafter puncturing an ear vein. The anesthesia is introduced through thecatheter by providing 3-5 mg of thiopental (Trapanal®) per killogram ofbody weight. After endotracheal intubation, the animals are ventilatedusing oxygen (30%), nitrous oxide (laughing gas) and isoflurane(Isofluran®). During the entire surgery, the analgesic fentanyldihydrogen citrate (Fentanyl®) having a dosage 0.2-0.4 mg, isadministered. At the same time, relaxation is achieved by administrationof atracurium besilate (Atracurium®) at a dosage of 0.5 mg/kg of bodyweight.

[0240] After complete exposure of the pedicles of lumbar vertebralbodies L4 to L6, a bilateral instrumentation of the pedicles L4 and L6takes place. This is performed by using pedicle screws of 5 mm or 6 mmin diameter, depending on the diameter found in the pedicles.Subsequently, a bilateral transpedicular removal of the disc of thecranial motor segment L4/L5 is performed over the pedicle of L5 underpediculoscopic control. The endplates of the affected vertebral bodiesare decorticated.

[0241] Inter- and intracorporal application of test samples occurs via atranspedicular cannula in all 12 sheep of the trial group. Test samplesinclude porous β-TCP, osteogenic protein or osteogenic proteinencapsulated in PLGA in varying concentrations. In the first controlgroup that consists of 12 sheep, only the porous β-TCP is applied. Inthe second control group, autologous spongiosa is administered insteadof the composition of this invention.

[0242] Finally, the internal fixator is installed completely. The typeof the internal fixator as well as the necessary instrumentation andsurgical procedure is standardized and well known to the skilledpractitioner. Drains are placed and the wound is closed using absorbablesuture for fascia and subcutis as well as skin staples.

[0243] During the entire surgical procedure, an x-ray image amplifier isavailable for intraoperative fluoroscopy. This facilitates exactorientation during the execution of the above steps.

[0244] Harvesting of the 12 sheep administered with autologous spongiosais carried out under anesthesia as follows: the left iliac crest skinand fascia is cut by making a longitudinal incision about 8 cm long. Thegluteal muscles are moved subperiostally and the cancellous bone graftis harvested from the iliac crest after an osteotomy. Bleeding controland placement of a Drain is performed upon closure of the wound inlayers. The harvesting procedure is standard and known to an ordinaryperson skilled in the art

[0245] Clinical Observations

[0246] Daily neurologic examinations are performed to evaluate the gaitof the animals as well as neurological deficits that may occurpostoperatively. Operative wounds are closely examined each day. Bodyweights are measured preoperatively and at the time of euthanasia.

[0247] Radiographic Analysis

[0248] Before evaluation, the complete lumbar spine is freshlydissected, and the internal fixator is carefully removed.Anteroposterior and plain lateral radiographs of the operated spinalsegments are obtained under consistent conditions of milliamperes,kilovolts, and seconds at 0 and 8 weeks to assist in fusion evaluation.The status of the fusion are evaluated with use of the grading systemdocumented by Lenke et al., J. Spinal Disord, 5, pp. 433-442 (1992),incorporated herein by reference. With this system, A indicates a big,solid trabeculated bilateral fusion mass (definitely solid); B, a big,solid unilateral fusion mass with a small contralateral fusion mass(possibly solid); C, a small, thin bilateral fusion mass with anapparent crack (probably not solid); and D, bilateral resorption of thegraft or fusion mass with an obvious bilateral pseudarthrosis(definitely not solid).

[0249] Additionally, computerized tomography scans are performed toassess the fusion mass in cross sections and in saggital-planereconstructions. For each fusion mass, approximately forty sequentialcomputerized tomography scans are made with use of two-millimeter sliceintervals and subsequent reconstruction in the saggital plane underconsistent magnification and radiographic conditions.

[0250] Biomechanical Testing

[0251] Four specimens of each group are evaluated biomechanically. Afterradiographic analysis, all muscles are carefully removed whilemaintaining the ligamentous and bony structures. The spines are frozenat −20° C. For each of these specimens, the upper half of the uppervertebra and the lower half of the lower vertebra of the motion segmentL4/L5 are embedded in polymethylmethacrylate (Technovit 3040; HeraeusKulzer GmbH, Wehrheim/Ts, Germany). Each specimen is then fixed andtested without preload in a spine tester in a non-destructive testingmode. Alternating sequences of flexion/extension, axial right/leftrotation, and right/left lateral bending moments are appliedcontinuously at a constant rate of 1.7 degrees/second by stepper motorsintegrated in the gimbal of the spine tester. Two precycles are appliedto minimize the effect of the viscous component in the viscoelasticresponse, and data are collected on the third cyle. Range of motion,neutral zone, and two stiffness parameters are determined from theresulting load-deformation curves.

[0252] Histology/Histomorphometry

[0253] Eight specimens of each group are evaluated histologically aftertwo, four or eight weeks postoperatively. After radiographic analysis,the spines are fixed in 10% formalin-solution. Cross sections of eitherspecimen are obtained to evaluate bony fusion, cellular reactions,biocompatibility, and signs of cement-integration/degradation.Qualitative histologic assessment of the fusion mass at the operativesite are made for the presence of giant cells, inflammatory cells, orfibrous responses where the implanted materials may have beenencapsulated. In addition, the osteoid found within the trabecularfusion mass and the amount of trabecular bone are assessed.Histomorphometric variables, such as the percentage of osteoid, osteoidthickness, number of osteoblasts per millimeter bone surface, and numberof osteoclasts per millimeter bone surface are determined.

[0254] Fluorochrome Labeling

[0255] Eight animals are subjected to intravenuous application of 90milligrams of xylenol orange per kilogram of body weight two weekspostoperatively, 10 milligrams of calcein green per kilogram of bodyweight four weeks postoperatively, and 25 milligrams ofdoxycyclinhyclate yellow per kilogram of body weight six weekspostoperatively. This regimen follows the method published by Rahn andPerren. See, e.g., Rahn et al., Stain Technology, 46, pp. 125-129(1971); Rahn et al., Akt Traumatol, 10, pp. 109-115 (1980). Fluorochromesequential analysis is then performed by Fluorescence microscopy on thespecimens under UV light for qualitative and quantitative dynamicevaluation.

EXAMPLE 11 Repair of Osteochondral Defects in Dogs

[0256] A total of 12 adult male dogs are utilized. Bilateralosteochondral defects, 5.0 mm in diameter and 6 mm deep, penetrating thesubchondral bone, are created in the central load bearing region of eachmedial femoral condyle. In 6 animals, the right defects will receive thehigh dose OP-1 encapsulated in PLGA. The left limb of all animals willreceive the collagen matrix plus CMC to serve as a control. Theremaining 6 dogs receive a low dose OP-1 encapsulated in PLGA on theright side and a control on the left side. The animals are sacrificed at16 weeks post-implantation. At sacrifice, the distal femurs areretrieved en bloc and the defect sites are evaluated histologically andgrossly based on the scheme of Moran et al., J. Bone Joint Surg. 74B,pp. 659-667 (1992), which is incorporated herein by reference.

[0257] Using standard aseptic techniques, surgery is performed underisofluorane gas anesthesia and the animals are monitored byelectrocardiogram and heart rate monitors. Pre-surgical medication isadministered approximately 20-30 minutes prior to anesthesia induction.The presurgical medication will consist of butorphanol tartrate (0.05mg/kg body weight). Anesthesia is administered by intravenous injectionof sodium pentothal (17.5 mg/kg body weight). Following induction, anendotracheal tube is placed and anesthesia is maintained by isofluoraneinhalation. Surgery is performed by making a medial parapatellarincision approximately 4 cm in length. The patella is retractedlaterally to expose the femoral condyle. In the right medial condyle, a5.0 mm diameter defect extending through the cartilage layer andpenetrating the subchondral bone to a depth of 6 mm is created with aspecially designed or modified 5.0 mm drill bit. After copiousirrigation with saline to remove bone debris and spilled marrow cells,the appropriate concentration of OP-1 encapsulated in PLGA is carefullypacked into each defect site with a blunt probe and by hand. Asufficient amount of OP-1 is placed within the defect so that it willflush with the articulating surface. While protecting the implantedmaterial, the joint is irrigated to remove any implant not placed withinthe defects. The joint capsule and soft-tissues are then meticulouslyclosed in layers with resorbable suture. The procedure is repeated onthe contralateral side with placement of a control.

[0258] Butorphanol tartrate (0.05 mg/kg body weight) is administeredsubcutaneously as required. Animals are administered intramuscularantibiotics for four days post-surgery and routine anterior-posteriorradiographs are taken immediately after surgery to insure propersurgical placement. Animals are kept in 3×4 feet recovery cages untilthe animal is able to tolerate weight bearing. Then, the animals aretransferred to runs and allowed unrestricted motion.

[0259] Radiographs of the hindlimbs are obtained preoperatively,immediately postoperative, and at 16 weeks (sacrifice). The preoperativeradiographs are used to assure that no pre-existing abnormalities arepresent and to verify skeletal maturity. Postoperative radiographs areused to assess defect placement. Sacrifice radiographs are used toassess the rate of healing and restoration of the subchondral bone andthe articulating surface. Radiographs are obtained within one week ofthe evaluation date.

[0260] At the appropriate time, animals are sacrificed using anintravenous barbiturate overdose. The distal femurs are immediatelyharvested en bloc and stored in saline soaked towels, placed in plasticbags labeled with the animal number, right or left designation, and anyother necessary identifiers. High power photographs of the defect sitesare taken and carefully labeled. Prior to sacrifice venous blood isdrawn for routine blood count with cell differential. Soft tissues aremeticulously dissected away from the defect site. The proximal end ofthe femur is removed. All specimens are prepared for histologicevaluation immediately after gross grading and photography. On a watercooled diamond saw each defect site is isolated.

[0261] The gross appearance of the defect sites and repair tissue isgraded based upon the study of Moran et al., supra. Points areapportioned according to the presence of intra-articular adhesions,restoration of the articular surface, cartilage erosion and appearance.

[0262] The individual specimens are fixed by immersion in 4%paraformaldehyde solution and prepared for decalcified histologicprocessing. Three sections from three levels are cut from each block.Levels 1 and 3 are closest to the defect perimeter. Level 2 is locatedat the defect center. Three sections from each level may be stained withtoluidine blue and Safranin 0 and fast green. Sections are graded basedupon the scheme of Moran et al., supra. This analysis apportions pointsbased upon the nature of the repair tissue, structural characteristics,and cellular changes. Descriptive statistics are calculated for grossand histologic parameters.

[0263] While we have described a number of embodiments of thisinvention, it is apparent that our basic constructions may be altered toprovide other embodiments which utilize the products and processes ofthis invention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims, rather than by thespecific embodiments which have been presented by way of example.

1 2001-03-02 1 431 PRT Homo sapiens 1 Met His Val Arg Ser Leu Arg AlaAla Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp Ala Pro Leu Phe LeuLeu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp Asn Glu Val His SerSer Phe Ile His Arg Arg Leu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met GlnArg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro HisLeu Gln Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met Phe Met Leu Asp LeuTyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln GlyPhe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 Thr Gln Gly Pro ProLeu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125 Asp Ala Asp MetVal Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140 Glu Phe PheHis Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150 155 160 SerLys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile 165 170 175Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile 180 185190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu 195200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn ProArg 225 230 235 240 His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu AspGly Gln Ser 245 250 255 Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg HisGly Pro Gln Asn 260 265 270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys AlaThr Glu Val His Phe 275 280 285 Arg Ser Ile Arg Ser Thr Gly Ser Lys GlnArg Ser Gln Asn Arg Ser 290 295 300 Lys Thr Pro Lys Asn Gln Glu Ala LeuArg Met Ala Asn Val Ala Glu 305 310 315 320 Asn Ser Ser Ser Asp Gln ArgGln Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp LeuGly Trp Gln Asp Trp Ile Ile Ala Pro Glu 340 345 350 Gly Tyr Ala Ala TyrTyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr Met AsnAla Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375 380 Phe Ile AsnPro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385 390 395 400 LeuAsn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 4302 96 PRT Artificial Sequence Description of Artificial SequenceSynthetic amino acid sequence 2 Leu Tyr Val Asp Phe Ser Asp Val Gly TrpAsp Asp Trp Ile Val Ala 1 5 10 15 Pro Pro Gly Tyr Gln Ala Phe Tyr CysHis Gly Glu Cys Pro Phe Pro 20 25 30 Leu Ala Asp His Phe Asn Ser Thr AsnHis Ala Val Val Gln Thr Leu 35 40 45 Val Asn Ser Val Asn Ser Lys Ile ProLys Ala Cys Cys Val Pro Thr 50 55 60 Glu Leu Ser Ala Ile Ser Met Leu TyrLeu Asp Glu Asn Glu Lys Val 65 70 75 80 Val Leu Lys Tyr Asn Gln Glu MetVal Val Glu Gly Cys Gly Cys Arg 85 90 95 3 96 PRT Artificial SequenceDescription of Artificial Sequence Synthetic amino acid sequence 3 LeuTyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala 1 5 10 15Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro 20 25 30Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Val Val Gln Thr Leu 35 40 45Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr 50 55 60Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val 65 70 7580 Val Leu Lys Tyr Asn Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg 85 9095 4 102 PRT Artificial Sequence Description of Artificial Sequence OPX4 Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 1 5 1015 Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 2530 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 35 4045 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 50 5560 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65 7075 80 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 8590 95 Xaa Ala Cys Gly Cys His 100 5 97 PRT Artificial SequenceDescription of Artificial Sequence Generic Sequence 7 5 Leu Xaa Xaa XaaPhe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Pro Xaa XaaXaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 50 55 60 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Val XaaLeu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90 95 Xaa 6102 PRT Artificial Sequence Description of Artificial Sequence GenericSequence 8 6 Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly TrpXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr CysXaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa AsnHis Ala 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa LeuXaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa XaaMet Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 7 97 PRT ArtificialSequence Description of Artificial Sequence Generic Sequence 9 7 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 ProXaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 55 60 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 95Xaa 8 102 PRT Artificial Sequence Description of Artificial SequenceGeneric Sequence 10 8 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa XaaXaa Xaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 9 5 PRTArtificial Sequence Description of Artificial Sequence Consensussequence 9 Cys Xaa Xaa Xaa Xaa 1 5 10 1822 DNA Homo sapiens CDS(49)..(1341) 10 ggtgcgggcc cggagcccgg agcccgggta gcgcgtagag ccggcgcg atgcac gtg 57 Met His Val 1 cgc tca ctg cga gct gcg gcg ccg cac agc ttc gtggcg ctc tgg gca 105 Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val AlaLeu Trp Ala 5 10 15 ccc ctg ttc ctg ctg cgc tcc gcc ctg gcc gac ttc agcctg gac aac 153 Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser LeuAsp Asn 20 25 30 35 gag gtg cac tcg agc ttc atc cac cgg cgc ctc cgc agccag gag cgg 201 Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser GlnGlu Arg 40 45 50 cgg gag atg cag cgc gag atc ctc tcc att ttg ggc ttg ccccac cgc 249 Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro HisArg 55 60 65 ccg cgc ccg cac ctc cag ggc aag cac aac tcg gca ccc atg ttcatg 297 Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met70 75 80 ctg gac ctg tac aac gcc atg gcg gtg gag gag ggc ggc ggg ccc ggc345 Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 8590 95 ggc cag ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag ggc393 Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly 100105 110 115 ccc cct ctg gcc agc ctg caa gat agc cat ttc ctc acc gac gccgac 441 Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp120 125 130 atg gtc atg agc ttc gtc aac ctc gtg gaa cat gac aag gaa ttcttc 489 Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe135 140 145 cac cca cgc tac cac cat cga gag ttc cgg ttt gat ctt tcc aagatc 537 His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile150 155 160 cca gaa ggg gaa gct gtc acg gca gcc gaa ttc cgg atc tac aaggac 585 Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp165 170 175 tac atc cgg gaa cgc ttc gac aat gag acg ttc cgg atc agc gtttat 633 Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr180 185 190 195 cag gtg ctc cag gag cac ttg ggc agg gaa tcg gat ctc ttcctg ctc 681 Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe LeuLeu 200 205 210 gac agc cgt acc ctc tgg gcc tcg gag gag ggc tgg ctg gtgttt gac 729 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val PheAsp 215 220 225 atc aca gcc acc agc aac cac tgg gtg gtc aat ccg cgg cacaac ctg 777 Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His AsnLeu 230 235 240 ggc ctg cag ctc tcg gtg gag acg ctg gat ggg cag agc atcaac ccc 825 Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile AsnPro 245 250 255 aag ttg gcg ggc ctg att ggg cgg cac ggg ccc cag aac aagcag ccc 873 Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys GlnPro 260 265 270 275 ttc atg gtg gct ttc ttc aag gcc acg gag gtc cac ttccgc agc atc 921 Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe ArgSer Ile 280 285 290 cgg tcc acg ggg agc aaa cag cgc agc cag aac cgc tccaag acg ccc 969 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser LysThr Pro 295 300 305 aag aac cag gaa gcc ctg cgg atg gcc aac gtg gca gagaac agc agc 1017 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu AsnSer Ser 310 315 320 agc gac cag agg cag gcc tgt aag aag cac gag ctg tatgtc agc ttc 1065 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr ValSer Phe 325 330 335 cga gac ctg ggc tgg cag gac tgg atc atc gcg cct gaaggc tac gcc 1113 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu GlyTyr Ala 340 345 350 355 gcc tac tac tgt gag ggg gag tgt gcc ttc cct ctgaac tcc tac atg 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu AsnSer Tyr Met 360 365 370 aac gcc acc aac cac gcc atc gtg cag acg ctg gtccac ttc atc aac 1209 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val HisPhe Ile Asn 375 380 385 ccg gaa acg gtg ccc aag ccc tgc tgt gcg ccc acgcag ctc aat gcc 1257 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr GlnLeu Asn Ala 390 395 400 atc tcc gtc ctc tac ttc gat gac agc tcc aac gtcatc ctg aag aaa 1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val IleLeu Lys Lys 405 410 415 tac aga aac atg gtg gtc cgg gcc tgt ggc tgc cactagctcctcc 1351 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425430 gagaattcag accctttggg gccaagtttt tctggatcct ccattgctcg ccttggccag1411 gaaccagcag accaactgcc ttttgtgaga ccttcccctc cctatcccca actttaaagg1471 tgtgagagta ttaggaaaca tgagcagcat atggcttttg atcagttttt cagtggcagc1531 atccaatgaa caagatccta caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac1591 gcataaagaa aaatggccgg gccaggtcat tggctgggaa gtctcagcca tgcacggact1651 cgtttccaga ggtaattatg agcgcctacc agccaggcca cccagccgtg ggaggaaggg1711 ggcgtggcaa ggggtgggca cattggtgtc tgtgcgaaag gaaaattgac ccggaagttc1771 ctgtaataaa tgtcacaata aaacgaatga atgaaaaaaa aaaaaaaaaa a 1822

We claim:
 1. A porous β-TCP comprising a porous body of beta-tricalciumphosphate comprising a multiplicity of pores, wherein the pores aresingle separate voids having a pore diameter size of 20-500 μm.
 2. Aporous β-TCP comprising a porous body of beta-tricalcium phosphatecomprising a multiplicity of pores, wherein the pores are singleseparate voids having a pore diameter size of 410-460 μm.
 3. A porousβ-TCP comprising a porous body of beta-tricalcium phosphate comprising amultiplicity of pores, wherein the pores are single separate voidshaving a pore diameter size of 40-190 μm.
 4. A porous β-TCP comprising aporous body of beta-tricalcium phosphate comprising a multiplicity ofpores, wherein the pores are single separate voids having a porediameter size of 20-95 μm.
 5. A porous β-TCP comprising a porous body ofbeta-tricalcium phosphate comprising a multiplicity of pores, whereinthe pores are single separate voids having a pore diameter size of50-125 μm.
 6. The porous β-TCP of any one of claims 1 to 5, wherein thebeta-tricalcium phosphate i s sintered.
 7. The porous β-TCP of any oneof claims 1 to 5, wherein the β-TCP is granular and has a particle sizeof 0.1-2 mm.
 8. The porous β-TCP of any one of claims 1 to 5, whereinthe β-TCP is granular and has a particle size of 0.5-1.7 mm.
 9. Theporous β-TCP of any one of claims 1 to 5, wherein the β-TCP is granularand has a particle size of 1-1.7 mm.
 10. The porous β-TCP of any one ofclaims 1 to 5, wherein the β-TCP is granular and has a particle size of0.5-1.0 mm.
 11. The porous β-TCP of any one of claims 1 to 5, whereinthe total porosity is in the range of 5-80%.
 12. The porous β-TCP of anyone of claims 1 to 5, wherein the total porosity is in the range of40-80%.
 13. The porous β-TCP of any one of claims 1 to 5, wherein thetotal porosity is in the range of 65-75%.
 14. The porous β-TCP of anyone of claims 1 to 5, wherein the total porosity is 70%.
 15. The porousβ-TCP of any one of claims 1 to 5, further comprising a bioactive agent.16. The porous β-TCP of claim 15, wherein the bioactive agent is a bonemorphogenic protein.
 17. The porous β-TCP of claim 16, wherein the bonemorphogenic protein is selected from the group consisting of OP-1, OP-2,OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b,BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6,GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121, dorsalin-1, DPP,Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, TGF-β andconservative amino acid sequence variants thereof having osteogenicactivity.
 18. The porous β-TCP of claim 15, wherein the bioactive agentis an osteogenic protein comprising an amino acid sequence having atleast 70% homology with the C-terminal 102-106 amino acids of humanOP-1.
 19. The porous β-TCP of claim 16 further comprising a morphogenicprotein stimulatory factor.
 20. The porous β-TCP of claim 19, whereinthe morphogenic protein stimulatory factor is selected from the groupconsisting of insulin-like growth factor I (IGF-I), estradiol,fibroblast growth factor (FGF), growth hormone (GH), growth anddifferentiation factor (GDF), hydrocortisone (HC), insulin,progesterone, parathyroid hormone (PTH), vitamin D, retinoic acid andIL-6.
 21. The porous β-TCP of claim 15, wherein the bioactive agent is anucleic acid molecule comprising a sequence encoding a bone morphogenicprotein.
 22. The porous β-TCP of claim 15, wherein the bioactive agentis encapsulated in a biodegradable agent.
 23. The porous β-TCP of claim22, wherein the biodegradable agent is selected from the groupconsisting of ethylenevinylacetate, natural and synthetic collagen,poly(glaxanone), poly(phosphazenes), polyglactin, polyglactic acid,polyaldonic acid, polyacrylic acids, polyalkanoates, polyorthoesters,poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(ζ-caprolactone),poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate) poly(D,L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more α-hydroxy carboxylic acid monomers, bioactiveglass compositions, admixtures thereof and any derivatives andmodifications thereof.
 24. The porous β-TCP of claim 23, wherein thePLGA has a molecular weight of 5 kD to 500 kD.
 25. The porous β-TCP ofclaim 23, wherein the PLGA has a molecular weight of 10 kD to 30 kD. 26.The porous β-TCP of claim 15, wherein the bioactive agent is anallograft or autograft.
 27. A moldable putty composition comprising theporous β-TCP according to any one of claims 1 to 5 and a binder.
 28. Themoldable putty composition of claim 27, wherein the binder is selectedfrom the group consisting of sodium alginate, hyaluronic acid, sodiumhyaluronate, gelatin, collagen, peptides, mucin, chrondroitin sulfate,chitosan, poloxamer, glycosaminoglycan, polysaccharide, polyethyleneglycol, methylcellulose, carboxy methylcellulose, carboxy 8methylcellulose sodium, carboxy methylcellulose calcium, hydroxypropylmethylcellulose, hydroxybutyl methylcellulose, hydroxyethylmethylcellulose, hydroxyethylcellulose, methylhydroxyethyl cellulose,hydroxyethyl cellulose, polylactic acid, polyglycolic acid, co-polymersof polylactic acid and polyglycolic acid, polyhydroxybutyric acid,polymalic acid, polyglutamic acid, polylactone, mannitol, whitepetrolatum, mannitol/dextran combinations, mannitol/white petrolatumcombinations, sesame oil, fibrin glue and admixtures thereof.
 29. Themoldable putty composition of claim 28, wherein the fibrin glue is amixture of human fibrinogen and thrombin.
 30. The moldable puttycomposition of claim 27 further comprising a bioactive agent.
 31. A kitcomprising: a) the porous β-TCP of any one of claims 1 to 5; and b) abioactive agent.
 32. The kit of claim 31, wherein the bioactive agent isa bone morphogenic protein.
 33. The kit of claim 32, wherein the bonemorphogenic protein is selected from the group consisting of OP-1, OP-2,OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b,BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6,GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121, dorsalin-1, DPP,Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, TGF-β andconservative amino acid sequence variants thereof having osteogenicactivity.
 34. The kit of claim 31, wherein the bioactive agent is anosteogenic protein comprising an amino acid sequence having at least 70%homology with the C-terminal 102-106 amino acids of human OP-1.
 35. Thekit of claim 32 further comprising a morphogenic protein stimulatoryfactor.
 36. The kit of claim 35, wherein the morphogenic proteinstimulatory factor is selected from the group consisting of insulin-likegrowth factor I (IGF-I), estradiol, fibroblast growth factor (FGF),growth hormone (GH), growth and differentiation factor (GDF),hydrocortisone (HC), insulin, progesterone, parathyroid hormone (PTH),vitamin D, retinoic acid and IL-6.
 37. A kit comprising: a) the porousβ-TCP of any one of claims 1 to 5; and b) a binder.
 38. The kit of claim37, wherein the binder is selected from the group consisting of sodiumalginate, hyaluronic acid, sodium hyaluronate, gelatin, collagen,peptides, mucin, chrondroitin sulfate, chitosan, poloxamer,glycosaminoglycan, polysaccharide, polyethylene glycol, methylcellulose,carboxy methylcellulose, carboxy methylcellulose sodium, carboxymethylcellulose calcium, hydroxypropyl methylcellulose, hydroxybutylmethylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose,methylhydroxyethyl cellulose, hydroxyethyl cellulose, polylactic acid,polyglycolic acid, co-polymers of polylactic acid and polyglycolic acid,polyhydroxybutyric acid, polymalic acid, polyglutamic acid, polylactone,mannitol, white petrolatum, mannitol/dextran combinations,mannitol/white petrolatum combinations, sesame oil, fibrin glue andadmixtures thereof.
 39. The kit of claim 38, wherein the fibrin glue isa mixture of human fibrinogen and thrombin.
 40. An implantableprosthetic device comprising: a) a prosthetic implant having a surfaceregion implantable adjacent to a target tissue; and b) the porous β-TCPof any one of claims 1 to 5 disposed on the surface region.
 41. Theprosthetic device of claim 40 further comprising a bioactive agentdispersed in the porous β-TCP.
 42. The prosthetic device of claim 41,wherein the bioactive agent is a bone morphogenic protein.
 43. Theprosthetic device of claim 42, wherein the bone morphogenic protein isselected from the group consisting of OP-1, OP-2, OP-3, COP-1, COP-3,COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6,BMP-9, BMP-10, BMP-1l, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17,BMP-18, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11, GDF-12, MP121, dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL,UNIVIN, SCREW, ADMP, NEURAL, TGF-β and conservative amino acid sequencevariants thereof having osteogenic activity.
 44. The prosthetic deviceof claim 41, wherein the bioactive agent is an osteogenic proteincomprising an amino acid sequence having at least 70% homology with theC-terminal 102-106 amino acids of human OP-1.
 45. The prosthetic deviceof claim 42 further comprising a morphogenic protein stimulatory factor.46. The prosthetic device of claim 45, wherein the morphogenic proteinstimulatory factor is selected from the group consisting of insulin-likegrowth factor I (IGF-I), estradiol, fibroblast growth factor (FGF),growth hormone (GH), growth and differentiation factor (GDF),hydrocortisone (HC), insulin, progesterone, parathyroid hormone (PTH),vitamin D, retinoic acid and IL-6.
 47. The prosthetic device of claim41, wherein the bioactive agent is a nucleic acid molecule comprising asequence encoding a bone morphogenic protein.
 48. The prosthetic deviceof claim 41, wherein the bioactive agent is encapsulated in abiodegradable agent.
 49. The prosthetic device of claim 48, wherein thebiodegradable agent is selected from the group consisting ofethylenevinylacetate, natural and synthetic collagen, poly(glaxanone),poly(phosphazenes), polyglactin, polyglactic acid, polyaldonic acid,polyacrylic acids, polyalkanoates, polyorthoesters, poly(L-lactide)(PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(ζ-caprolactone),poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate) poly(D,L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more α-hydroxy carboxylic acid monomers, bioactiveglass compositions, admixtures thereof and any derivatives andmodifications thereof.
 50. The prosthetic device of claim 49, whereinthe PLGA has a molecular weight of 5 kD to 500 kD.
 51. The prostheticdevice of claim 49, wherein the PLGA has a molecular weight of 10 kD to30 kD.
 52. The prosthetic device of claim 40, wherein the device isselected from the group consisting of a hip device, a fusion cage and amaxillofacial device.
 53. The prosthetic device of claim 40 furthercomprising a binder.
 54. The prosthetic device of claim 53, wherein thebinder is selected from the group consisting of sodium alginate,hyaluronic acid, sodium hyaluronate, gelatin, collagen, peptides, mucin,chrondroitin sulfate, chitosan, poloxamer, glycosaminoglycan,polysaccharide, polyethylene glycol, methylcellulose, carboxymethylcellulose, carboxy methylcellulose sodium, carboxy methylcellulosecalcium, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose,hydroxyethyl methylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxyethyl cellulose, polylactic acid, polyglycolic acid,co-polymers of polylactic acid and polyglycolic acid, polyhydroxybutyricacid, polymalic acid, polyglutamic acid, polylactone, mannitol, whitepetrolatum, mannitol/dextran combinations, mannitol/white petrolatumcombinations, sesame oil, fibrin glue and admixtures thereof.
 55. Theprosthetic device of claim 54, wherein the fibrin glue is a mixture ofhuman fibrinogen and thrombin.
 56. A method of producing a porous β-TCPgranule comprising: (a) blending a TCP powder with a pore-forming agent;(b) adding a granulating solution to form a crumbly mass; (c) passingthe crumbly mass through a sieve to form granules; and (d) sintering thegranules to form porous β-TCP.
 57. A method of producing a porous β-TCPgranule comprising: (a) blending a TCP powder with a pore-forming agent,wherein the proportion of pore-forming agent is 37.5% by weight; (b)adding a granulating solution to form a crumbly mass; (c) passing thecrumbly mass through a sieve to form granules; and (d) sintering thegranules to form porous β-TCP.
 58. A method of producing a porous β-TCPgranule comprising: (a) blending a TCP powder with a pore-forming agent;(b) adding a granulating solution to form a crumbly mass; (c) passingthe crumbly mass through a sieve to form granules, wherein the sieve isin the size range of 500-1000 μm or 1000-1700 μm; and (d) sintering thegranules to form porous β-TCP.
 59. A method of producing a porous β-TCPgranule comprising: (a) blending a TCP powder with a pore-forming agent;(b) adding a granulating solution to form a crumbly mass; (c) passingthe crumbly mass through a sieve to form granules; (d) vaporizing thegranules at 700-800° C.; and (e) sintering the granules to form porousβ-TCP.
 60. A method of producing a porous β-TCP granule comprising: (a)blending a TCP powder with a pore-forming agent; (b) adding agranulating solution to form a crumbly mass; (c) passing the crumblymass through a sieve to form granules; and (d) sintering the granules at1000-1200° C. and followed by a slow cooling step to form porous β-TCP.61. The method of any one of claims 56 to 60, wherein the pore-formingagent is selected from the group consisting of prepolymers ofpolyacrylates, polymethacrylates, polymethyl methacrylate, copolymers ofmethyl acrylate and methyl methacrylate, polystyrene, polyethyleneglycol, crystalline cellulose, fibrous cellulose, polyurethanes,polyethylenes, nylon resins and acrylic resins.
 62. The method of anyone of claims 56 to 60, wherein the granulating solution comprises acompound selected from the group consisting of polyvinyl pyrrolidone,starch, gelatin, polyvinyl alcohol, polyethylene oxide, hydroxyethylcellulose, polyvinyl butyral and cellulose acetate butyrate.
 63. Themethod of any one of claims 56 to 60, wherein the porous β-TCP isresieved after formation.
 64. A composition comprising tricalciumphosphate powder and a pore-forming agent, wherein the pore-formingagent has a diameter of 20-500 μm.
 65. A composition comprisingtricalcium phosphate powder and a pore-forming agent, wherein thepore-forming agent has a diameter of 410-460 μm.
 66. A compositioncomprising tricalcium phosphate powder and a pore-forming agent, whereinthe pore-forming agent has a diameter of 40-190 μm.
 67. A compositioncomprising tricalcium phosphate powder and a pore-forming agent, whereinthe pore-forming agent has a diameter of 20-95 μm.
 68. A compositioncomprising tricalcium phosphate powder and a pore-forming agent, whereinthe pore-forming agent has a diameter of 50-125 μm.
 69. The compositionof any one of claims 64 to 68, wherein the proportion of pore-formingagent is 30-40% by weight.
 70. A method of inducing bone formation in amammal comprising the step of implanting in the defect site of saidmammal a composition comprising the porous β-TCP according to any one ofclaims 1 to
 5. 71. The method of claim 70, wherein the compositionfurther comprises a bioactive agent.
 72. The method of claim 71, whereinthe bioactive agent is a bone morphogenic protein.
 73. The method ofclaim 70, wherein the composition further comprises a binder.
 74. Amethod of delivering a bioactive agent at a site requiring boneformation comprising implanting at the defect site of a mammal acomposition comprising the porous β-TCP of any one of claims 1 to 5 anda bioactive agent.
 75. The method of claim 74, wherein the bioactiveagent is a bone morphogenic protein.
 76. The method of claim 74, whereinthe bioactive agent is encapsulated in a biodegradable agent.
 77. Themethod of claim 76, wherein the delivery of the bioactive agent issustained released.
 78. The method of claim 74, wherein the bioactiveagent is a nucleic acid molecule comprising a sequence encoding a bonemorphogenic protein.
 79. A method of delivering a bioactive agent at asite requiring cartilage formation comprising implanting at the defectsite of a mammal a composition comprising a bioactive agent and abiodegradable agent having a particle size of 20-500 μm, wherein thebioactive agent is encapsulated in the biodegradable agent.
 80. Acomposition comprising a bioactive agent encapsulated in a biodegradableagent, wherein the biodegradable agent has a particle size of 20-500 μm.81. A composition comprising a bioactive agent encapsulated in abiodegradable agent, wherein the biodegradable agent has a particle sizeof 20-140 μm.
 82. A composition comprising a bioactive agentencapsulated in a biodegradable agent, wherein the biodegradable agenthas a particle size of 75-140 μm.
 83. The composition of any one ofclaims 80 to 82, where in the biodegradable agent is selected from thegroup consisting of ethylenevinylacetate, natural and syntheticcollagen, poly(glaxanone), poly(phosphazenes), polyglactin, polyglacticacid, polyaldonic acid, polyacrylic acids, polyalkanoates,polyorthoesters, poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA),polyglycolide (PGA), poly(lactide-co-glycolide (PLGA),poly(ζ-caprolactone), poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate) poly(D,L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more α-hydroxy carboxylic acid monomers, bioactiveglass compositions, admixtures thereof and any derivatives andmodifications thereof.
 84. The composition of claim 83, wherein the PLGAhas a molecular weight of 5 kD to 500 kD.
 85. The composition of claim83, wherein the PLGA has a molecular weight of 10 kD to 30 kD.